Methods, compositions, and kits comprising linker probes for quantifying polynucleotides

ABSTRACT

The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3′ target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3′ target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/009,681,filed Jan. 28, 2016, now U.S. Pat. No. 9,657,346, which is acontinuation of U.S. application Ser. No. 13/612,485, filed Sep. 12,2012, now abandoned, which is a continuation of U.S. application Ser.No. 12/543,466, filed Aug. 18, 2009, now U.S. Pat. No. 9,068,222, whichis a continuation of U.S. application Ser. No. 10/947,460, filed Sep.21, 2004, now U.S. Pat. No. 7,575,863, which claims the benefit of U.S.Provisional Application 60/575,661, filed May 28, 2004, each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 6, 2009, is named533USC1.txt, and is 118,670 bytes in size.

FIELD

The present teachings are in the field of molecular and cell biology,specifically in the field of detecting target polynucleotides such asmiRNA.

INTRODUCTION

RNA interference (RNAi) is a highly coordinated, sequence-specificmechanism involved in posttranscriptional gene regulation. During theinitial steps of process, a ribonuclease (RNase) II-like enzyme calledDicer reduces long double-strand RNA (dsRNA) and complex hairpinprecursors into: 1) small interfering RNAs (siRNA) that degrademessenger RNA (mRNA) and 2) micro RNAs (miRNAs) that can target mRNAsfor cleavage or attenuate translation.

The siRNA class of molecules is thought to be comprised of 21-23nucleotide (nt) duplexes with characteristic dinucleotide 3′ overhangs(Ambros et al., 2003, RNA, 9 (3), 277-279). siRNA has been shown to actas the functional intermediate in RNAi, specifically directing cleavageof complementary mRNA targets in a process that is commonly regarded tobe an antiviral cellular defense mechanism (Elbashir et al., 2001,Nature, 411:6836), 494-498, Elbashir et al., 2001, Genes andDevelopment, 15 (2), 188-200). Target RNA cleavage is catalyzed by theRNA-induced silencing complex (RISC), which functions as a siRNAdirected endonuclease (reviewed in Bartel, 2004, Cell, 116 (2),281-297).

Micro RNAs (miRNAs) typically comprise single-stranded, endogenousoligoribonucleotides of roughly 22 (18-25) bases in length that areprocessed from larger stem-looped precursor RNAs. The first genesrecognized to encode miRNAs, lin-4 and let-7 of C. elegans, wereidentified on the basis of the developmental timing defects associatedwith the loss-of-function mutations (Lee et al., 1993, Cell, 75 (5),843-854; Reinhart et al., 2000, Nature, 403, (6772), 901-906; reviewedby Pasquinelli et al., 2002, Annual Review of Cell and DevelopmentalBiology, 18, 495-513). The breadth and importance of miRNA-directed generegulation are coming into focus as more miRNAs and regulatory targetsand functions are discovered. To date, a total of at least 700 miRNAshave been identified in C. elegans, Drosophila (Fire et al., 1998,Nature, 391 (6669(805-811), mouse, human (Lagos-Quintana et al., 2001,Science, 294 (5543), 853-858), and plants (Reinhart et al., 2002, Genesand Development, 16 (13), 1616-1626). Their sequences are typicallyconserved among different species. Size ranges from 18 to 25 nucleotidesfor miRNAs are the most commonly observed to date.

The function of most miRNAs is not known. Recently discovered miRNAfunctions include control of cell proliferation, cell death, and fatmetabolism in flies (Brennecke et al., 2003, cell, 113 (1), 25-36; Xu etal, 2003, Current Biology, 13 (9), 790-795), neuronal patterning innematodes (Johnston and Hobert, 2003, Nature, 426 (6968), 845-849),modulation of hematopoietic lineage differentiation in mammals (Chen etal., 2004, Science, 303 (5654), 83-87), and control of leaf and flowerdevelopment in plants (Aukerman and Sakai, 2003, Plant Cell, 15 (11),2730-2741; Chen, 2003, Science, 303 (5666):2022-2025; Emery et al.,2003, Current Biology, 13 (20), 1768-1774; Palatnik et al., 2003,Nature, 425 (6955), 257-263). There is speculation that miRNAs mayrepresent a new aspect of gene regulation.

Most miRNAs have been discovered by cloning. There are few cloning kitsavailable for researchers from Ambion and QIAGEN etc. The process islaborious and less accurate. Further, there has been little reliabletechnology available for miRNA quantitation (Allawi et al., Third WaveTechnologies, R N A. 2004 July; 10(7):1153-61). Northern blotting hasbeen used but results are not quantitative (Lagos-Quitana et al., 2001,Science, 294 (5543), 853-854). Many miRNA researchers are interested inmonitoring the level of the miRNAs at different tissues, at thedifferent stages of development, or after treatment with variouschemical agents. However, the short length of miRNAs has their studydifficult.

SUMMARY

In some embodiments, the present teachings provide a method fordetecting a micro RNA (miRNA) comprising; hybridizing the miRNA and alinker probe, wherein the linker probe comprises a stem, a loop, and a3′ target-specific portion, wherein the 3′ target-specific portion basepairs with the 3′ end region of the miRNA; extending the linker probe toform an extension reaction product; amplifying the extension reactionproduct to form an amplification product; and, detecting the miRNA.

In some embodiments, the present teachings provide a method fordetecting a target polynucleotide comprising; hybridizing the targetpolynucleotide and a linker probe, wherein the linker probe comprises astem, a loop, and a 3′ target-specific portion, wherein the 3′target-specific portion base pairs with the 3′ end region of the targetpolynucleotide; extending the linker probe to form an extension reactionproduct; amplifying the extension reaction product to form anamplification product in the presence of a detector probe, wherein thedetector probe comprises a nucleotide of the linker probe stem in theamplification product or a nucleotide of the linker probe stemcomplement in the amplification product; and, detecting the targetpolynucleotide.

In some embodiments, the present teachings provide a method fordetecting a miRNA molecule comprising; hybridizing the miRNA moleculeand a linker probe, wherein the linker probe comprises a stem, a loop,and a 3′ target specific portion, wherein the 3′ target-specific portionbase pairs with the 3′ end region of the target polynucleotide;extending the linker probe to form an extension reaction product;amplifying the extension reaction product in the presence of a detectorprobe to form an amplification product, wherein the detector probecomprises a nucleotide of the linker probe stem in the amplificationproduct or a nucleotide of the linker probe stem complement in theamplification product, and the detector probe further comprises anucleotide of the 3′ end region of the miRNA in the amplificationproduct or a nucleotide of the 3′ end region of the miRNA complement inthe amplification product; and, detecting the miRNA molecule.

In some embodiments, the present teachings provide a method fordetecting two different miRNAs from a single hybridization reactioncomprising; hybridizing a first miRNA and a first linker probe, and asecond miRNA and a second linker probe, wherein the first linker probeand the second linker probe each comprise a loop, a stem, and a 3′target-specific portion, wherein the 3′ target-specific portion of thefirst linker probe base pairs with the 3′ end region of the first miRNA,and wherein the 3′ target-specific portion of the second linker probebase pairs with the 3′ end region of the second miRNA; extending thefirst linker probe and the second linker probe to form extensionreaction products; dividing the extension reaction products into a firstamplification reaction to form a first amplification reaction product,and a second amplification reaction to form a second amplificationreaction product, wherein a primer in the first amplification reactioncorresponds with the first miRNA and not the second miRNA, and a primerin the second amplification reaction corresponds with the second miRNAand not the first miRNA, wherein a first detector probe in the firstamplification reaction differs from a second detector probe in thesecond amplification reaction, wherein the first detector probecomprises a nucleotide of the first linker probe stem of theamplification product or a nucleotide of the first linker probe stemcomplement in the first amplification product, wherein the seconddetector probe comprises a nucleotide of the second linker probe stem ofthe amplification product or a nucleotide of the second linker probestem complement in the amplification product; and, detecting the twodifferent miRNAs.

In some embodiments, the present teachings provide a method fordetecting two different target polynucleotides from a singlehybridization reaction comprising; hybridizing a first targetpolynucleotide and a first linker probe, and a second targetpolynucleotide and a second linker probe, wherein the first linker probeand the second linker probe each comprise a loop, a stem, and a 3′target-specific portion, wherein the 3′ target-specific portion of thefirst linker probe base pairs with the 3′ end region of the first targetpolynucleotide, and wherein the 3′ target-specific portion of the secondlinker probe base pairs with the 3′ end region of the second targetpolynucleotide; extending the first linker probe and the second linkerprobe to form extension reaction products; dividing the extensionreaction products into a first amplification reaction to form a firstamplification reaction product and a second amplification reaction toform a second amplification reaction product; and, detecting the twodifferent miRNA molecules.

In some embodiments, the present teachings provide a method fordetecting a miRNA molecule from a cell lysate comprising; hybridizingthe miRNA molecule from the cell lysate with a linker probe, wherein thelinker probe comprises a stem, a loop, and a 3′ target specific portion,wherein the 3′ target-specific portion base pairs with the 3′ end regionof the miRNA; extending the linker probe to form an extension reactionproduct; amplifying the extension reaction product to form anamplification product in the presence of a detector probe, wherein thedetector probe comprises a nucleotide of the linker probe stem of theamplification product or a nucleotide of the linker probe stemcomplement in the amplification product, and the detector probe furthercomprises a nucleotide of the 3′ end region of the miRNA in theamplification product or a nucleotide of the 3′ end region of the miRNAcomplement in the amplification product; and, detecting the miRNAmolecule.

A kit comprising; a reverse transcriptase and a linker probe, whereinthe linker probe comprises a stem, a loop, and a 3′ target-specificportion, wherein the 3′ target-specific portion corresponds to a miRNA.

The present teachings contemplate method for detecting a miRNA moleculecomprising a step of hybridizing, a step of extending, a step ofamplifying, and a step of detecting.

These and other features of the present teachings are set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIGS. 1A, 1B, and 1C depict certain aspects of various compositionsaccording to some embodiments of the present teachings.

FIGS. 2A, 2B, 2C, and 2D depict certain aspects of various compositionsaccording to some embodiments of the present teachings.

FIG. 3 depicts certain sequences of various compositions according tosome embodiments of the present teachings. FIG. 3 depicts SEQ ID No.780, the oligonucleotide for the micro RNA MiR-16 (boxed, 11) and alinker probe (13).

FIG. 4 depicts one single-plex assay design according to someembodiments of the present teachings.

FIG. 5 depicts an overview of a multiplex assay design according to someembodiments of the present teachings.

FIG. 6 depicts a multiplex assay design according to some embodiments ofthe present teachings.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way. The section headings usedherein are for organizational purposes only and are not to be construedas limiting the described subject matter in any way. All literature andsimilar materials cited in this application, including but not limitedto, patents, patent applications, articles, books, treatises, andinternet web pages are expressly incorporated by reference in theirentirety for any purpose. When definitions of terms in incorporatedreferences appear to differ from the definitions provided in the presentteachings, the definition provided in the present teachings shallcontrol. It will be appreciated that there is an implied “about” priorto the temperatures, concentrations, times, etc discussed in the presentteachings, such that slight and insubstantial deviations are within thescope of the present teachings herein. In this application, the use ofthe singular includes the plural unless specifically stated otherwise.For example, “a primer” means that more than one primer can, but neednot, be present; for example but without limitation, one or more copiesof a particular primer species, as well as one or more versions of aparticular primer type, for example but not limited to, a multiplicityof different forward primers. Also, the use of “comprise”, “comprises”,“comprising”, “contain”, “contains”, “containing”, “include”,“includes”, and “including” are not intended to be limiting. It is to beunderstood that both the foregoing general description and the followingdetailed description are exemplary and explanatory only and are notrestrictive of the invention.

Some Definitions

As used herein, the term “target polynucleotide” refers to apolynucleotide sequence that is sought to be detected. The targetpolynucleotide can be obtained from any source, and can comprise anynumber of different compositional components. For example, the targetcan be nucleic acid (e.g. DNA or RNA), transfer RNA, siRNA, and cancomprise nucleic acid analogs or other nucleic acid mimic. The targetcan be methylated, non-methylated, or both. The target can bebisulfite-treated and non-methylated cytosines converted to uracil.Further, it will be appreciated that “target polynucleotide” can referto the target polynucleotide itself, as well as surrogates thereof, forexample amplification products, and native sequences. In someembodiments, the target polynucleotide is a miRNA molecule. In someembodiments, the target polynucleotide lacks a poly-A tail. In someembodiments, the target polynucleotide is a short DNA molecule derivedfrom a degraded source, such as can be found in for example but notlimited to forensics samples (see for example Butler, 2001, Forensic DNATyping: Biology and Technology Behind STR Markers. The targetpolynucleotides of the present teachings can be derived from any of anumber of sources, including without limitation, viruses, prokaryotes,eukaryotes, for example but not limited to plants, fungi, and animals.These sources may include, but are not limited to, whole blood, a tissuebiopsy, lymph, bone marrow, amniotic fluid, hair, skin, semen,biowarfare agents, anal secretions, vaginal secretions, perspiration,saliva, buccal swabs, various environmental samples (for example,agricultural, water, and soil), research samples generally, purifiedsamples generally, cultured cells, and lysed cells. It will beappreciated that target polynucleotides can be isolated from samplesusing any of a variety of procedures known in the art, for example theApplied Biosystems ABI Prism™ 6100 Nucleic Acid PrepStation, and the ABIPrism™ 6700 Automated Nucleic Acid Workstation, Boom et al., U.S. Pat.No. 5,234,809, mirVana RNA isolation kit (Ambion), etc. It will beappreciated that target polynucleotides can be cut or sheared prior toanalysis, including the use of such procedures as mechanical force,sonication, restriction endonuclease cleavage, or any method known inthe art. In general, the target polynucleotides of the present teachingswill be single stranded, though in some embodiments the targetpolynucleotide can be double stranded, and a single strand can resultfrom denaturation.

As used herein, the term “3′ end region of the target polynucleotide”refers to the region of the target to which the 3′ target specificportion of the linker probe hybridizes. In some embodiments there can bea gap between the 3′ end region of the target polynucleotide and the 5′end of the linker probe, with extension reactions filling in the gap,though generally such scenarios are not preferred because of the likelydestabilizing effects on the duplex. In some embodiments, a miRNAmolecule is the target, in which case the term “3′ end region of themiRNA” is used.

As used herein, the term “linker probe” refers to a molecule comprisinga 3′ target specific portion, a stem, and a loop. Illustrative linkerprobes are depicted in FIGS. 2A-2D and elsewhere in the presentteachings. It will be appreciated that the linker probes, as well as theprimers of the present teachings, can be comprised of ribonucleotides,deoxynucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, modified phosphate-sugar-backboneoligonucleotides, nucleotide analogs, or combinations thereof. For someillustrative teachings of various nucleotide analogs etc, see Fasman,1989, Practical Handbook of Biochemistry and Molecular Biology, pp.385-394, CRC Press, Boca Raton, Fla., Loakes, N. A. R. 2001, vol29:2437-2447, and Pellestor et al., Int J Mol Med. 2004 April;13(4):521-5.), references cited therein, and recent articles citingthese reviews. It will be appreciated that the selection of the linkerprobes to query a given target polynucleotide sequence, and theselection of which collection of target polynucleotide sequences toquery in a given reaction with which collection of linker probes, willinvolve procedures generally known in the art, and can involve the useof algorithms to select for those sequences with minimal secondary andtertiary structure, those targets with minimal sequence redundancy withother regions of the genome, those target regions with desirablethermodynamic characteristics, and other parameters desirable for thecontext at hand.

As used herein, the term “3′ target-specific portion” refers to thesingle stranded portion of a linker probe that is complementary to atarget polynucleotide. The 3′ target-specific portion is locateddownstream from the stem of the linker probe. Generally, the 3′target-specific portion is between 6 and 8 nucleotides long. In someembodiments, the 3′ target-specific portion is 7 nucleotides long. Itwill be appreciated that routine experimentation can produce otherlengths, and that 3′ target-specific portions that are longer than 8nucleotides or shorter than 6 nucleotides are also contemplated by thepresent teachings. Generally, the 3′-most nucleotides of the 3′target-specific portion should have minimal complementarity overlap, orno overlap at all, with the 3′ nucleotides of the forward primer; itwill be appreciated that overlap in these regions can produce undesiredprimer dimer amplification products in subsequent amplificationreactions. In some embodiments, the overlap between the 3′-mostnucleotides of the 3′ target-specific portion and the 3′ nucleotides ofthe forward primer is 0, 1, 2, or 3 nucleotides. In some embodiments,greater than 3 nucleotides can be complementary between the 3′-mostnucleotides of the 3′ target-specific portion and the 3′ nucleotides ofthe forward primer, but generally such scenarios will be accompanied byadditional non-complementary nucleotides interspersed therein. In someembodiments, modified bases such as LNA can be used in the 3′ targetspecific portion to increase the Tm of the linker probe (see for examplePetersen et al., Trends in Biochemistry (2003), 21:2:74-81). In someembodiments, universal bases can be used, for example to allow forsmaller libraries of linker probes. Universal bases can also be used inthe 3′ target specific portion to allow for the detection of unknowntargets. For some descriptions of universal bases, see for exampleLoakes et al., Nucleic Acids Research, 2001, Volume 29, No. 12,2437-2447. In some embodiments, modifications including but not limitedto LNAs and universal bases can improve reverse transcriptionspecificity and potentially enhance detection specificity.

As used herein, the term “stem” refers to the double stranded region ofthe linker probe that is between the 3′ target-specific portion and theloop. Generally, the stem is between 6 and 20 nucleotides long (that is,6-20 complementary pairs of nucleotides, for a total of 12-40 distinctnucleotides). In some embodiments, the stem is 8-14 nucleotides long. Asa general matter, in those embodiments in which a portion of thedetector probe is encoded in the stem, the stem can be longer. In thoseembodiments in which a portion of the detector probe is not encoded inthe stem, the stem can be shorter. Those in the art will appreciate thatstems shorter that 6 nucleotides and longer than 20 nucleotides can beidentified in the course of routine methodology and without undueexperimentation, and that such shorter and longer stems are contemplatedby the present teachings. In some embodiments, the stem can comprise anidentifying portion.

As used herein, the term “loop” refers to a region of the linker probethat is located between the two complementary strands of the stem, asdepicted in FIGS. 1A-1C and elsewhere in the present teachings.Typically, the loop comprises single stranded nucleotides, though othermoieties modified DNA or RNA, Carbon spacers such as C18, and/or PEG(polyethylene glycol) are also possible. Generally, the loop is between4 and 20 nucleotides long. In some embodiments, the loop is between 14and 18 nucleotides long. In some embodiments, the loop is 16 nucleotideslong. As a general matter, in those embodiments in which a reverseprimer is encoded in the loop, the loop can generally be longer. Inthose embodiments in which the reverse primer corresponds to both thetarget polynucleotide as well as the loop, the loop can generally beshorter. Those in the art will appreciate that loops shorter that 4nucleotides and longer than 20 nucleotides can be identified in thecourse of routine methodology and without undue experimentation, andthat such shorter and longer loops are contemplated by the presentteachings. In some embodiments, the loop can comprise an identifyingportion.

As used herein, the term “identifying portion” refers to a moiety ormoieties that can be used to identify a particular linker probe species,and as a result determine a target polynucleotide sequence, and canrefer to a variety of distinguishable moieties including zipcodes, aknown number of nucleobases, and combinations thereof. In someembodiments, an identifying portion, or an identifying portioncomplement, can hybridize to a detector probe, thereby allowingdetection of a target polynucleotide sequence in a decoding reaction.The terms “identifying portion complement” typically refers to at leastone oligonucleotide that comprises at least one sequence of nucleobasesthat are at least substantially complementary to and hybridize withtheir corresponding identifying portion. In some embodiments,identifying portion complements serve as capture moieties for attachingat least one identifier portion:element complex to at least onesubstrate; serve as “pull-out” sequences for bulk separation procedures;or both as capture moieties and as pull-out sequences (see for exampleO'Neil, et al., U.S. Pat. Nos. 6,638,760, 6,514,699, 6,146,511, and6,124,092). Typically, identifying portions and their correspondingidentifying portion complements are selected to minimize: internal,self-hybridization; cross-hybridization with different identifyingportion species, nucleotide sequences in a reaction composition,including but not limited to gDNA, different species of identifyingportion complements, or target-specific portions of probes, and thelike; but should be amenable to facile hybridization between theidentifying portion and its corresponding identifying portioncomplement. Identifying portion sequences and identifying portioncomplement sequences can be selected by any suitable method, for examplebut not limited to, computer algorithms such as described in PCTPublication Nos. WO 96/12014 and WO 96/41011 and in European PublicationNo. EP 799,897; and the algorithm and parameters of SantaLucia (Proc.Natl. Acad. Sci. 95:1460-65 (1998)). Descriptions of identifyingportions can be found in, among other places, U.S. Pat. No. 6,309,829(referred to as “tag segment” therein); U.S. Pat. No. 6,451,525(referred to as “tag segment” therein); U.S. Pat. No. 6,309,829(referred to as “tag segment” therein); U.S. Pat. No. 5,981,176(referred to as “grid oligonucleotides” therein); U.S. Pat. No.5,935,793 (referred to as “identifier tags” therein); and PCTPublication No. WO 01/92579 (referred to as “addressablesupport-specific sequences” therein). In some embodiments, the stem ofthe linker probe, the loop of the linker probe, or combinations thereofcan comprise an identifying portion, and the detector probe canhybridize to the corresponding identifying portion. In some embodiments,the detector probe can hybridize to both the identifying portion as wellas sequence corresponding to the target polynucleotide. In someembodiments, at least two identifying portion: identifying portioncomplement duplexes have melting temperatures that fall within a ΔT_(m)range (T_(max)−T_(min)) of no more than 10° C. of each other. In someembodiments, at least two identifying portion: identifying portioncomplement duplexes have melting temperatures that fall within a ΔT_(m)range of 5° C. or less of each other. In some embodiments, at least twoidentifying portion: identifying portion complement duplexes havemelting temperatures that fall within a ΔT_(m) range of 2° C. or less ofeach other. In some embodiments, at least one identifying portion or atleast one identifying portion complement is used to separate the elementto which it is bound from at least one component of a ligation reactioncomposition, a digestion reaction composition, an amplified ligationreaction composition, or the like. In some embodiments, identifyingportions are used to attach at least one ligation product, at least oneligation product surrogate, or combinations thereof, to at least onesubstrate. In some embodiments, at least one ligation product, at leastone ligation product surrogate, or combinations thereof, comprise thesame identifying portion. Examples of separation approaches include butare not limited to, separating a multiplicity of different element:identifying portion species using the same identifying portioncomplement, tethering a multiplicity of different element: identifyingportion species to a substrate comprising the same identifying portioncomplement, or both. In some embodiments, at least one identifyingportion complement comprises at least one label, at least one mobilitymodifier, at least one label binding portion, or combinations thereof.In some embodiments, at least one identifying portion complement isannealed to at least one corresponding identifying portion and,subsequently, at least part of that identifying portion complement isreleased and detected, see for example Published P.C.T. ApplicationWO04/4634 to Rosenblum et al., and Published P.C.T. ApplicationWO01/92579 to Wenz et al.,

As used herein, the term “extension reaction” refers to an elongationreaction in which the 3′ target specific portion of a linker probe isextended to form an extension reaction product comprising a strandcomplementary to the target polynucleotide. In some embodiments, thetarget polynucleotide is a miRNA molecule and the extension reaction isa reverse transcription reaction comprising a reverse transcriptase. Insome embodiments, the extension reaction is a reverse transcriptionreaction comprising a polymerase derived from a Eubacteria. In someembodiments, the extension reaction can comprise rTth polymerase, forexample as commercially available from Applied Biosystems catalog numberN808-0192, and N808-0098. In some embodiments, the target polynucleotideis a miRNA or other RNA molecule, and as such it will be appreciatedthat the use of polymerases that also comprise reverse transcriptionproperties can allow for some embodiments of the present teachings tocomprise a first reverse transcription reaction followed thereafter byan amplification reaction, thereby allowing for the consolidation of tworeactions in essentially a single reaction. In some embodiments, thetarget polynucleotide is a short DNA molecule and the extension reactioncomprises a polymerase and results in the synthesis of a 2^(nd) strandof DNA. In some embodiments, the consolidation of the extension reactionand a subsequent amplification reaction is further contemplated by thepresent teachings.

As used herein, the term “primer portion” refers to a region of apolynucleotide sequence that can serve directly, or by virtue of itscomplement, as the template upon which a primer can anneal for any of avariety of primer nucleotide extension reactions known in the art (forexample, PCR). It will be appreciated by those of skill in the art thatwhen two primer portions are present on a single polynucleotide, theorientation of the two primer portions is generally different. Forexample, one PCR primer can directly hybridize to a first primerportion, while the other PCR primer can hybridize to the complement ofthe second primer portion. In addition, “universal” primers and primerportions as used herein are generally chosen to be as unique as possiblegiven the particular assays and host genomes to ensure specificity ofthe assay.

As used herein, the term “forward primer” refers to a primer thatcomprises an extension reaction product portion and a tail portion. Theextension reaction product portion of the forward primer hybridizes tothe extension reaction product. Generally, the extension reactionproduct portion of the forward primer is between 9 and 19 nucleotides inlength. In some embodiments, the extension reaction product portion ofthe forward primer is 16 nucleotides. The tail portion is locatedupstream from the extension reaction product portion, and is notcomplementary with the extension reaction product; after a round ofamplification however, the tail portion can hybridize to complementarysequence of amplification products. Generally, the tail portion of theforward primer is between 5-8 nucleotides long. In some embodiments, thetail portion of the forward primer is 6 nucleotides long. Those in theart will appreciate that forward primer tail portion lengths shorterthan 5 nucleotides and longer than 8 nucleotides can be identified inthe course of routine methodology and without undue experimentation, andthat such shorter and longer forward primer tail portion lengths arecontemplated by the present teachings. Further, those in the art willappreciate that lengths of the extension reaction product portion of theforward primer shorter than 9 nucleotides in length and longer than 19nucleotides in length can be identified in the course of routinemethodology and without undue experimentation, and that such shorter andlonger extension reaction product portion of forward primers arecontemplated by the present teachings.

As used herein, the term “reverse primer” refers to a primer that whenextended forms a strand complementary to the target polynucleotide. Insome embodiments, the reverse primer corresponds with a region of theloop of the linker probe. Following the extension reaction, the forwardprimer can be extended to form a second strand product. The reverseprimer hybridizes with this second strand product, and can be extendedto continue the amplification reaction. In some embodiments, the reverseprimer corresponds with a region of the loop of the linker probe, aregion of the stem of the linker probe, a region of the targetpolynucleotide, or combinations thereof. Generally, the reverse primeris between 13-16 nucleotides long. In some embodiments the reverseprimer is 14 nucleotides long. In some embodiments, the reverse primercan further comprise a non-complementary tail region, though such a tailis not required. In some embodiments, the reverse primer is a “universalreverse primer,” which indicates that the sequence of the reverse primercan be used in a plurality of different reactions querying differenttarget polynucleotides, but that the reverse primer nonetheless is thesame sequence.

The term “upstream” as used herein takes on its customary meaning inmolecular biology, and refers to the location of a region of apolynucleotide that is on the 5′ side of a “downstream” region.Correspondingly, the term “downstream” refers to the location of aregion of a polynucleotide that is on the 3′ side of an “upstream”region.

As used herein, the term “hybridization” refers to the complementarybase-pairing interaction of one nucleic acid with another nucleic acidthat results in formation of a duplex, triplex, or other higher-orderedstructure, and is used herein interchangeably with “annealing.”Typically, the primary interaction is base specific, e.g., A/T and G/C,by Watson/Crick and Hoogsteen-type hydrogen bonding. Base-stacking andhydrophobic interactions can also contribute to duplex stability.Conditions for hybridizing detector probes and primers to complementaryand substantially complementary target sequences are well known, e.g.,as described in Nucleic Acid Hybridization, A Practical Approach, B.Hames and S. Higgins, eds., IRL Press, Washington, D.C. (1985) and J.Wetmur and N. Davidson, Mol. Biol. 31:349 et seq. (1968). In general,whether such annealing takes place is influenced by, among other things,the length of the polynucleotides and the complementary, the pH, thetemperature, the presence of mono- and divalent cations, the proportionof G and C nucleotides in the hybridizing region, the viscosity of themedium, and the presence of denaturants. Such variables influence thetime required for hybridization. Thus, the preferred annealingconditions will depend upon the particular application. Such conditions,however, can be routinely determined by the person of ordinary skill inthe art without undue experimentation. It will be appreciated thatcomplementarity need not be perfect; there can be a small number of basepair mismatches that will minimally interfere with hybridization betweenthe target sequence and the single stranded nucleic acids of the presentteachings. However, if the number of base pair mismatches is so greatthat no hybridization can occur under minimally stringent conditionsthen the sequence is generally not a complementary target sequence.Thus, complementarity herein is meant that the probes or primers aresufficiently complementary to the target sequence to hybridize under theselected reaction conditions to achieve the ends of the presentteachings.

As used herein, the term “amplifying” refers to any means by which atleast a part of a target polynucleotide, target polynucleotidesurrogate, or combinations thereof, is reproduced, typically in atemplate-dependent manner, including without limitation, a broad rangeof techniques for amplifying nucleic acid sequences, either linearly orexponentially. Exemplary means for performing an amplifying step includeligase chain reaction (LCR), ligase detection reaction (LDR), ligationfollowed by Q-replicase amplification, PCR, primer extension, stranddisplacement amplification (SDA), hyperbranched strand displacementamplification, multiple displacement amplification (MDA), nucleic acidstrand-based amplification (NASBA), two-step multiplexed amplifications,rolling circle amplification (RCA) and the like, including multiplexversions or combinations thereof, for example but not limited to,OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (alsoknown as combined chain reaction—CCR), and the like. Descriptions ofsuch techniques can be found in, among other places, Sambrook et al.Molecular Cloning, 3^(rd) Edition; Ausbel et al.; PCR Primer: ALaboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); TheElectronic Protocol Book, Chang Bioscience (2002), Msuih et al., J.Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R.Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., CurrOpin Biotechnol. 1993 February; 4(1):41-7, U.S. Pat. Nos. 6,027,998;6,605,451, Barany et al., PCT Publication No. WO 97/31256; Wenz et al.,PCT Publication No. WO 01/92579; Day et al., Genomics, 29(1): 152-162(1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCRProtocols: A Guide to Methods and Applications, Academic Press (1990);Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al.,Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development ofa Multiplex Ligation Detection Reaction DNA Typing Assay, SixthInternational Symposium on Human Identification, 1995 (available on theworld wide web at: promega.com/geneticidproc/ussymp6proc/blegrad.html);LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene,2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi andSambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. AcidRes. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66(2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl.Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18(2002);Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren etal., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002November; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May;53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February;12(1):21-7, U.S. Pat. Nos. 5,830,711, 6,027,889, 5,686,243, PublishedP.C.T. Application WO0056927A3, and Published P.C.T. ApplicationWO9803673A1. In some embodiments, newly-formed nucleic acid duplexes arenot initially denatured, but are used in their double-stranded form inone or more subsequent steps. An extension reaction is an amplifyingtechnique that comprises elongating a linker probe that is annealed to atemplate in the 5′ to 3′ direction using an amplifying means such as apolymerase and/or reverse transcriptase. According to some embodiments,with appropriate buffers, salts, pH, temperature, and nucleotidetriphosphates, including analogs thereof, i.e., under appropriateconditions, a polymerase incorporates nucleotides complementary to thetemplate strand starting at the 3′-end of an annealed linker probe, togenerate a complementary strand. In some embodiments, the polymeraseused for extension lacks or substantially lacks 5′ exonuclease activity.In some embodiments of the present teachings, unconventional nucleotidebases can be introduced into the amplification reaction products and theproducts treated by enzymatic (e.g., glycosylases) and/orphysical-chemical means in order to render the product incapable ofacting as a template for subsequent amplifications. In some embodiments,uracil can be included as a nucleobase in the reaction mixture, therebyallowing for subsequent reactions to decontaminate carryover of previousuracil-containing products by the use of uracil-N-glycosylase (see forexample Published P.C.T. Application WO9201814A2). In some embodimentsof the present teachings, any of a variety of techniques can be employedprior to amplification in order to facilitate amplification success, asdescribed for example in Radstrom et al., Mol Biotechnol. 2004 February;26(2):133-46. In some embodiments, amplification can be achieved in aself-contained integrated approach comprising sample preparation anddetection, as described for example in U.S. Pat. Nos. 6,153,425 and6,649,378. Reversibly modified enzymes, for example but not limited tothose described in U.S. Pat. No. 5,773,258, are also within the scope ofthe disclosed teachings. The present teachings also contemplate variousuracil-based decontamination strategies, wherein for example uracil canbe incorporated into an amplification reaction, and subsequentcarry-over products removed with various glycosylase treatments (see forexample U.S. Pat. No. 5,536,649, and U.S. Provisional Application60/584,682 to Andersen et al.,). Those in the art will understand thatany protein with the desired enzymatic activity can be used in thedisclosed methods and kits. Descriptions of DNA polymerases, includingreverse transcriptases, uracil N-glycosylase, and the like, can be foundin, among other places, Twyman, Advanced Molecular Biology, BIOSScientific Publishers, 1999; Enzyme Resource Guide, rev. 092298,Promega, 1998; Sambrook and Russell; Sambrook et al.; Lehninger; PCR:The Basics; and Ausbel et al.

As used herein, the term “detector probe” refers to a molecule used inan amplification reaction, typically for quantitative or real-time PCRanalysis, as well as end-point analysis. Such detector probes can beused to monitor the amplification of the target polynucleotide. In someembodiments, detector probes present in an amplification reaction aresuitable for monitoring the amount of amplicon(s) produced as a functionof time. Such detector probes include, but are not limited to, the5′-exonuclease assay (TaqMan® probes described herein (see also U.S.Pat. No. 5,538,848) various stem-loop molecular beacons (see e.g., U.S.Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, NatureBiotechnology 14:303-308), stemless or linear beacons (see, e.g., WO99/21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., 2001,SPIE 4264:53-58), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097),Sunrise®/Amplifluor® probes (U.S. Pat. No. 6,548,250), stem-loop andduplex Scorpion™ probes (Solinas et al., 2001, Nucleic Acids Research29:E96 and U.S. Pat. No. 6,589,743), bulge loop probes (U.S. Pat. No.6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons(U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences),hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA)light-up probes, self-assembled nanoparticle probes, andferrocene-modified probes described, for example, in U.S. Pat. No.6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al.,1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, MolecularCell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35;Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002,Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, NucleicAcids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332;Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al.,2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem Res.Toxicol. 15:118-126; and Yu et al., 2001, J. Am. Chem. Soc14:11155-11161. Detector probes can also comprise quenchers, includingwithout limitation black hole quenchers (Biosearch), Iowa Black (IDT),QSY quencher (Molecular Probes), and Dabsyl and Dabcelsulfonate/carboxylate Quenchers (Epoch). Detector probes can alsocomprise two probes, wherein for example a fluor is on one probe, and aquencher is on the other probe, wherein hybridization of the two probestogether on a target quenches the signal, or wherein hybridization onthe target alters the signal signature via a change in fluorescence.Detector probes can also comprise sulfonate derivatives of fluorescenindyes with SO3 instead of the carboxylate group, phosphoramidite forms offluorescein, phosphoramidite forms of CY 5 (commercially available forexample from Amersham). In some embodiments, interchelating labels areused such as ethidium bromide, SYBR® Green I (Molecular Probes), andPicoGreen® (Molecular Probes), thereby allowing visualization inreal-time, or end point, of an amplification product in the absence of adetector probe. In some embodiments, real-time visualization cancomprise both an intercalating detector probe and a sequence-baseddetector probe can be employed. In some embodiments, the detector probeis at least partially quenched when not hybridized to a complementarysequence in the amplification reaction, and is at least partiallyunquenched when hybridized to a complementary sequence in theamplification reaction. In some embodiments, the detector probes of thepresent teachings have a Tm of 63-69 C, though it will be appreciatedthat guided by the present teachings routine experimentation can resultin detector probes with other Tms. In some embodiments, probes canfurther comprise various modifications such as a minor groove binder(see for example U.S. Pat. No. 6,486,308) to further provide desirablethermodynamic characteristics. In some embodiments, detector probes cancorrespond to identifying portions or identifying portion complements.

The term “corresponding” as used herein refers to a specificrelationship between the elements to which the term refers. Somenon-limiting examples of corresponding include: a linker probe cancorrespond with a target polynucleotide, and vice versa. A forwardprimer can correspond with a target polynucleotide, and vice versa. Alinker probe can correspond with a forward primer for a given targetpolynucleotide, and vice versa. The 3′ target-specific portion of thelinker probe can correspond with the 3′ region of a targetpolynucleotide, and vice versa. A detector probe can correspond with aparticular region of a target polynucleotide and vice versa. A detectorprobe can correspond with a particular identifying portion and viceversa. In some cases, the corresponding elements can be complementary.In some cases, the corresponding elements are not complementary to eachother, but one element can be complementary to the complement of anotherelement.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, Aft AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the term “reaction vessel” generally refers to anycontainer in which a reaction can occur in accordance with the presentteachings. In some embodiments, a reaction vessel can be an eppendorftube, and other containers of the sort in common practice in modernmolecular biology laboratories. In some embodiments, a reaction vesselcan be a well in microtitre plate, a spot on a glass slide, or a well inan Applied Biosystems TaqMan Low Density Array for gene expression(formerly MicroCard™). For example, a plurality of reaction vessels canreside on the same support. In some embodiments, lab-on-a-chip likedevices, available for example from Caliper and Fluidgm, can provide forreaction vessels. In some embodiments, various microfluidic approachesas described in U.S. Provisional Application 60/545,674 to Wenz et al.,can be employed. It will be recognized that a variety of reaction vesselare available in the art and within the scope of the present teachings.

As used herein, the term “detection” refers to any of a variety of waysof determining the presence and/or quantity and/or identity of a targetpolynucleotide. In some embodiments employing a donor moiety and signalmoiety, one may use certain energy-transfer fluorescent dyes. Certainnonlimiting exemplary pairs of donors (donor moieties) and acceptors(signal moieties) are illustrated, e.g., in U.S. Pat. Nos. 5,863,727;5,800,996; and 5,945,526. Use of some combinations of a donor and anacceptor have been called FRET (Fluorescent Resonance Energy Transfer).In some embodiments, fluorophores that can be used as signaling probesinclude, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5(Cy 5), fluorescein, Vic™ Liz™, Tamra™, 5-Fam™, 6-Fam™, and Texas Red(Molecular Probes). (Vic™ Liz™, Tamra™, 5-Fam™, and 6-Fam™ (allavailable from Applied Biosystems, Foster City, Calif.). In someembodiments, the amount of detector probe that gives a fluorescentsignal in response to an excited light typically relates to the amountof nucleic acid produced in the amplification reaction. Thus, in someembodiments, the amount of fluorescent signal is related to the amountof product created in the amplification reaction. In such embodiments,one can therefore measure the amount of amplification product bymeasuring the intensity of the fluorescent signal from the fluorescentindicator. According to some embodiments, one can employ an internalstandard to quantify the amplification product indicated by thefluorescent signal. See, e.g., U.S. Pat. No. 5,736,333. Devices havebeen developed that can perform a thermal cycling reaction withcompositions containing a fluorescent indicator, emit a light beam of aspecified wavelength, read the intensity of the fluorescent dye, anddisplay the intensity of fluorescence after each cycle. Devicescomprising a thermal cycler, light beam emitter, and a fluorescentsignal detector, have been described, e.g., in U.S. Pat. Nos. 5,928,907;6,015,674; and 6,174,670, and include, but are not limited to the ABIPrism® 7700 Sequence Detection System (Applied Biosystems, Foster City,Calif.), the ABI GeneAmp® 5700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.), the ABI GeneAmp® 7300 SequenceDetection System (Applied Biosystems, Foster City, Calif.), and the ABIGeneAmp® 7500 Sequence Detection System (Applied Biosystems). In someembodiments, each of these functions can be performed by separatedevices. For example, if one employs a Q-beta replicase reaction foramplification, the reaction may not take place in a thermal cycler, butcould include a light beam emitted at a specific wavelength, detectionof the fluorescent signal, and calculation and display of the amount ofamplification product. In some embodiments, combined thermal cycling andfluorescence detecting devices can be used for precise quantification oftarget nucleic acid sequences in samples. In some embodiments,fluorescent signals can be detected and displayed during and/or afterone or more thermal cycles, thus permitting monitoring of amplificationproducts as the reactions occur in “real time.” In some embodiments, onecan use the amount of amplification product and number of amplificationcycles to calculate how much of the target nucleic acid sequence was inthe sample prior to amplification. In some embodiments, one could simplymonitor the amount of amplification product after a predetermined numberof cycles sufficient to indicate the presence of the target nucleic acidsequence in the sample. One skilled in the art can easily determine, forany given sample type, primer sequence, and reaction condition, how manycycles are sufficient to determine the presence of a given targetpolynucleotide. As used herein, determining the presence of a target cancomprise identifying it, as well as optionally quantifying it. In someembodiments, the amplification products can be scored as positive ornegative as soon as a given number of cycles is complete. In someembodiments, the results may be transmitted electronically directly to adatabase and tabulated. Thus, in some embodiments, large numbers ofsamples can be processed and analyzed with less time and labor when suchan instrument is used. In some embodiments, different detector probesmay distinguish between different target polynucleotides. A non-limitingexample of such a probe is a 5′-nuclease fluorescent probe, such as aTaqMan® probe molecule, wherein a fluorescent molecule is attached to afluorescence-quenching molecule through an oligonucleotide link element.In some embodiments, the oligonucleotide link element of the 5′-nucleasefluorescent probe binds to a specific sequence of an identifying portionor its complement. In some embodiments, different 5′-nucleasefluorescent probes, each fluorescing at different wavelengths, candistinguish between different amplification products within the sameamplification reaction. For example, in some embodiments, one could usetwo different 5′-nuclease fluorescent probes that fluoresce at twodifferent wavelengths (WL_(A) and WL_(B)) and that are specific to twodifferent stem regions of two different extension reaction products (A′and B′, respectively). Amplification product A′ is formed if targetnucleic acid sequence A is in the sample, and amplification product B′is formed if target nucleic acid sequence B is in the sample. In someembodiments, amplification product A′ and/or B′ may form even if theappropriate target nucleic acid sequence is not in the sample, but suchoccurs to a measurably lesser extent than when the appropriate targetnucleic acid sequence is in the sample. After amplification, one candetermine which specific target nucleic acid sequences are present inthe sample based on the wavelength of signal detected and theirintensity. Thus, if an appropriate detectable signal value of onlywavelength WL_(A) is detected, one would know that the sample includestarget nucleic acid sequence A, but not target nucleic acid sequence B.If an appropriate detectable signal value of both wavelengths WL_(A) andWL_(B) are detected, one would know that the sample includes both targetnucleic acid sequence A and target nucleic acid sequence B. In someembodiments, detection can occur through any of a variety of mobilitydependent analytical techniques based on differential rates of migrationbetween different analyte species. Exemplary mobility-dependent analysistechniques include electrophoresis, chromatography, mass spectroscopy,sedimentation, e.g., gradient centrifugation, field-flow fractionation,multi-stage extraction techniques, and the like. In some embodiments,mobility probes can be hybridized to amplification products, and theidentity of the target polynucleotide determined via a mobilitydependent analysis technique of the eluted mobility probes, as describedfor example in Published P.C.T. Application WO04/46344 to Rosenblum etal., and WO01/92579 to Wenz et al., In some embodiments, detection canbe achieved by various microarrays and related software such as theApplied Biosystems Array System with the Applied Biosystems 1700Chemiluminescent Microarray Analyzer and other commercially availablearray systems available from Affymetrix, Agilent, Illumina, and AmershamBiosciences, among others (see also Gerry et al., J. Mol. Biol.292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; andStears et al., Nat. Med. 9:140-45, including supplements, 2003). It willalso be appreciated that detection can comprise reporter groups that areincorporated into the reaction products, either as part of labeledprimers or due to the incorporation of labeled dNTPs during anamplification, or attached to reaction products, for example but notlimited to, via hybridization tag complements comprising reporter groupsor via linker arms that are integral or attached to reaction products.Detection of unlabeled reaction products, for example using massspectrometry, is also within the scope of the current teachings.

EXEMPLARY EMBODIMENTS

FIGS. 1A-1C depict certain compositions according to some embodiments ofthe present teachings. FIG. 1A, a miRNA molecule (1, dashed line) isdepicted. FIG. 1B, a linker probe (2) is depicted, illustrating a 3′target specific portion (3), a stem (4), and a loop (5). FIG. 1C, amiRNA hybridized to a linker probe is depicted, illustrating the 3′target specific portion of the linker probe (3) hybridized to the 3′ endregion of the miRNA (6).

As shown in FIGS. 2A-2D, a target polynucleotide (9, dotted line) isillustrated to show the relationship with various components of thelinker probe (10), the detector probe (7), and the reverse primer (8),according to various non-limiting embodiments of the present teachings.For example as shown in FIG. 2A, in some embodiments the detector probe(7) can correspond with the 3′ end region of the target polynucleotidein the amplification product as well as a region upstream from the 3′end region of the target polynucleotide in the amplification product.(Here, the detector probe is depicted as rectangle (7) with an F and aQ, symbolizing a TaqMan probe with a fluorophore (F) and a quencher(Q)). Also shown in FIG. 2A, the loop can correspond to the reverseprimer (8). In some embodiments as shown in FIG. 2B, the detector probe(7) can correspond with a region of the amplification productcorresponding with the 3′ end region of the target polynucleotide in theamplification product, as well as a region upstream from the 3′ endregion of the target polynucleotide in the amplification product, aswell as the linker probe stem in the amplification product. Also shownin FIG. 2B, the upstream region of the stem, as well as the loop, cancorrespond to the reverse primer (8). In some embodiments as shown inFIG. 2C, the detector probe can correspond to the amplification productin a manner similar to that shown in FIG. 2B, but the loop cancorrespond to the reverse primer (8). In some embodiments as shown inFIG. 2D, the detector probe (7) can correspond with the linker probestem in the amplification product. Also shown in FIG. 2D, the upstreamregion of the stem, as well as the loop can correspond to the reverseprimer (8). It will be appreciated that various related strategies forimplementing the different functional regions of these compositions arepossible in light of the present teachings, and that such derivationsare routine to one having ordinary skill in the art without undueexperimentation.

FIG. 3 depicts the nucleotide relationship for the micro RNA MiR-16(boxed, 11) according to some embodiments of the present teachings.Shown here is the interrelationship of MiR-16 to a forward primer (12)(SEQ ID No. 781), a linker probe (13), a TaqMan detector probe (14) (SEQID No. 782), and a reverse primer (boxed, 15) (SEQ ID No. 783). TheTaqMan probe comprises a 3′ minor groove binder (MGB), and a 5′ FAMfluorophore. It will be appreciated that in some embodiments of thepresent teachings the detector probes, such as for example TaqManprobes, can hybridize to either strand of an amplification product. Forexample, in some embodiments the detector probe can hybridize to thestrand of the amplification product corresponding to the first strandsynthesized. In some embodiments, the detector probe can hybridize tothe strand of the amplification product corresponding to the secondstrand synthesized.

FIG. 4 depicts a single-plex assay design according to some embodimentsof the present teachings. Here, a miRNA molecule (16) and a linker probe(17) are hybridized together (18). The 3′ end of the linker probe of thetarget-linker probe composition is extended to form an extension product(19) that can be amplified in a PCR. The PCR can comprise a miRNAspecific forward primer (20) and a reverse primer (21). The detection ofa detector probe (22) during the amplification allows for quantitationof the miRNA.

FIG. 5 depicts an overview of a multiplex assay design according to someembodiments of the present teachings. Here, a multiplexed hybridizationand extension reaction is performed in a first reaction vessel (23).Thereafter, aliquots of the extension reaction products from the firstreaction vessel are transferred into a plurality of amplificationreactions (here, depicted as PCRs 1, 2, and 3) in a plurality of secondreaction vessels. Each PCR can comprise a distinct primer pair and adistinct detector probe. In some embodiments, a distinct primer pair butthe same detector probe can be present in each of a plurality of PCRs.

FIG. 6 depicts a multiplex assay design according to some embodiments ofthe present teachings. Here, three different miRNAs (24, 25, and 26) arequeried in a hybridization reaction comprising three different linkerprobes (27, 28, and 29). Following hybridization and extension to formextension products (30, 31, and 32), the extension products are dividedinto three separate amplification reactions. (Though not explicitlyshown, it will be appreciated that a number of copies of the moleculesdepicted by 30, 31, and 32 can be present, such that each of the threeamplification reactions can have copies of 30, 31, and 32.) PCR 1comprises a forward primer specific for miRNA 24 (33), PCR 2 comprises aforward primer specific for miRNA 25 (34), and PCR 3 comprises a forwardprimer specific for miRNA 26 (35). Each of the forward primers furthercomprise a non-complementary tail portion. PCR 1, PCR 2, and PCR 3 allcomprise the same universal reverse primer 36. Further, PCR 1 comprisesa distinct detector probe (37) that corresponds to the 3′ end region ofmiRNA 24 and the stem of linker probe 27, PCR 2 comprises a distinctdetector probe (38) that corresponds to the 3′ end region of miRNA 25and the stem of linker probe 28, and PCR 3 comprises a distinct detectorprobe (39) that corresponds to the 3′ region of miRNA 26 and the stem oflinker probe 29.

The present teachings also contemplate reactions comprisingconfigurations other than a linker probe. For example, in someembodiments, two hybridized molecules with a sticky end can be employed,wherein for example an overlapping 3′ sticky end hybridizes with the 3′end region of the target polynucleotide. Some descriptions of twomolecule configurations that can be employed in the present teachingscan be found in Chen et al., U.S. Provisional Application 60/517,470.Viewed in light of the present teachings herein, one of skill in the artwill appreciate that the approaches of Chen et al., can also be employedto result in extension reaction products that are longer that the targetpolynucleotide. These longer products can be detected with detectorprobes by, for example, taking advantage of the additional nucleotidesintroduced into the reaction products.

The present teachings also contemplate embodiments wherein the linkerprobe is ligated to the target polynucleotide, as described for examplein Chen et al., U.S. Provisional Application 60/575,661, and thecorresponding co-filed U.S. Provisional application co-filed herewith

Further, it will be appreciated that in some embodiments of the presentteachings, the two molecule configurations in Chen et al., U.S.Provisional Application 60/517,470 can be applied in embodimentscomprising the linker approaches discussed in Chen et al., U.S.Provisional Application 60/575,661.

Generally however, the loop structure of the present teachings willenhance the Tm of the target polynucleotide-linker probe duplex. Withoutbeing limited to any particular theory, this enhanced Tm could possiblybe due to base stacking effects. Also, the characteristics of the loopedlinker probe of the present teachings can minimize nonspecific primingduring the extension reaction, and/or a subsequent amplificationreaction such as PCR. Further, the looped linker probe of the presentteachings can better differentiate mature and precursor forms of miRNA,as illustrated infra in Example 6.

The present teachings also contemplate encoding and decoding reactionschemes, wherein a first encoding extension reaction is followed by asecond decoding amplification reaction, as described for example inLivak et al., U.S. Provisional Application 60/556,162, Chen et al., U.S.Provisional Application 60/556,157, Andersen et al., U.S. ProvisionalApplication 60/556,224, and Lao et al., U.S. Provisional Application60/556,163.

The present teachings also contemplate a variety of strategies tominimize the number of different molecules in multiplexed amplificationstrategies, as described for example in Whitcombe et al., U.S. Pat. No.6,270,967.

In certain embodiments, the present teachings also provide kits designedto expedite performing certain methods. In some embodiments, kits serveto expedite the performance of the methods of interest by assembling twoor more components used in carrying out the methods. In someembodiments, kits may contain components in pre-measured unit amounts tominimize the need for measurements by end-users. In some embodiments,kits may include instructions for performing one or more methods of thepresent teachings. In certain embodiments, the kit components areoptimized to operate in conjunction with one another.

For example, the present teachings provide a kit comprising, a reversetranscriptase and a linker probe, wherein the linker probe comprises astem, a loop, and a 3′ target-specific portion, wherein the 3′target-specific portion corresponds to a miRNA. In some embodiments, thekits can comprise a DNA polymerase. In some embodiments, the kits cancomprise a primer pair. In some embodiments, the kits can furthercomprise a forward primer specific for a miRNA, and, a universal reverseprimer, wherein the universal reverse primer comprises a nucleotide ofthe loop of the linker probe. In some embodiments, the kits can comprisea plurality of primer pairs, wherein each primer pair is in one reactionvessel of a plurality of reaction vessels. In some embodiments, the kitscan comprise a detector probe. In some embodiments, the detector probecomprises a nucleotide of the linker probe stem in the amplificationproduct or a nucleotide of the linker probe stem complement in theamplification product, and the detector probe further comprises anucleotide of the 3′ end region of the miRNA in the amplificationproduct or a nucleotide of the 3′ end region of the miRNA complement inthe amplification product.

The present teachings further contemplate kits comprising a means forhybridizing, a means for extending, a means for amplifying, a means fordetecting, or combinations thereof.

While the present teachings have been described in terms of theseexemplary embodiments, the skilled artisan will readily understand thatnumerous variations and modifications of these exemplary embodiments arepossible without undue experimentation. All such variations andmodifications are within the scope of the current teachings. Aspects ofthe present teachings may be further understood in light of thefollowing examples, which should not be construed as limiting the scopeof the teachings in any way.

Example 1

A single-plex reaction was performed in replicate for a collection ofmouse miRNAs, and the effect of the presence or absence of ligase, aswell as the presence or absence of reverse transcriptase, determined.The results are shown in Table 1 as Ct values.

First, a 6 ul reaction was set up comprising: 1 ul Reverse TranscriptionEnzyme Mix (Applied Biosystems part number 4340444) (or 1 ul dH2O), 0.5ul T4 DNA Ligase (400 units/ul, NEB) (or 0.5 ul dH20), 0.25 ul 2M KCl,0.05 ul dNTPs (25 mM each), 0.25 ul T4 Kinase (10 units/ul, NEB), 1 ul10×T4 DNA ligase buffer (NEB), 0.25 ul Applied Biosystems RNaseInhibitor (10 units/up, and 2.2 ul dH20 Next, 2 ul of the linker probe(0.25 uM) and RNA samples (2 ul of 0.25 ug/ul mouse lung total RNA(Ambion, product number 7818) were added. Next, the reaction was mixed,spun briefly, and placed on ice for 5 minutes.

The reaction was then incubated at 16 C for 30 minutes, 42 C for 30minutes, followed by 85 C for 5 minutes, and then held at 4 C. Thereactions were diluted 4 times by adding 30 ul of dH20 prior to the PCRamplification.

A 10 ul PCR amplification was then set up comprising: 2 ul of dilutedreverse transcription reaction product, 1.3 ul 10 uM miRNA specificForward Primer, 0.7 ul 10 uM Universal Reverse Primer, 0.2 ul TaqMandetector probe, 0.2 ul dNTPs (25 mM each), 0.6 ul dH20, 5 ul 2×TaqManmaster mix (Applied Biosystems, without UNG). The reaction was startedwith a 95 C step for 10 minutes. Then, 40 cycles were performed, eachcycle comprising 95 C for 15 seconds, and 60 C for 1 minute. Table 1indicates the results of this experiment.

TABLE 1 Reverse miRNA Replicate Ligase transcriptase Let-7a1 mir16 mir20mir21 mir26a mir30a mir224 average Yes Yes 16.8 16.0 19.1 16.8 15.0 21.327.3 18.9 Yes No 38.7 31.3 39.9 31.9 30.1 33.3 40.0 35.0 I No Yes 18.014.6 18.3 16.2 14.0 21.3 26.4 18.4 No No 40.0 36.6 40.0 40.0 33.8 39.240.0 38.5 Yes Yes 17.1 16.2 19.3 17.0 15.1 21.4 27.3 19.1 Yes No 38.931.2 37.6 32.1 30.4 33.4 39.4 34.7 II No Yes 18.4 14.8 18.7 16.6 14.321.5 26.7 18.7 No No 40.0 36.1 40.0 40.0 34.1 40.0 40.0 38.6 ReplicateYes Yes 16.9 16.1 19.2 16.9 15.0 21.4 27.3 19.0 Average Yes No 38.8 31.238.8 32.0 30.3 33.4 39.7 34.9 No Yes 18.2 14.7 18.5 16.4 14.1 21.4 26.618.6 No No 40.0 36.4 40.0 40.0 34.0 39.6 40.0 40.0

Sequences of corresponding forward primers, reverse primer, and TaqManprobes are shown in Table 2.

TABLE 2 SEQ ID miRNA ID NO: miRNA sequences miR-16 1uagcagcacguaaauauuggcg miR-20 2 uaaagugcuuauagugcaggua miR-21 3uagcuuaucagacugauguuga miR-22 4 aagcugccaguugaagaacugu miR-26a 5uucaaguaauccaggauaggcu miR-29 6 cuagcaccaucugaaaucgguu miR-30a 7cuuucagucggauguuugcagc miR-34 8 uggcagugucuuagcugguugu miR-200b 9cucuaauacugccugguaaugaug miR-323 10 gcacauuacacggucgaccucu miR-324-5 11cgcauccccuagggcauuggugu Let-7a1 12 ugagguaguagguuguauaguu SEQ IDLinker probe NO: Linker probe sequences miR-16linR6 13GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACCGCCAA miR20LinR6 14GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACTACCTG miR-21linR6 15GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACTCAACA miR-22linR6 16GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACACAGTT miR-26alinR6 17GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACAGCCTA miR-29linR6 18GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACAACCGA miR30LinR6 19GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACGCTGCA miR-34linR6 20GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACACAACC miR-200blinR6 21GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACCATCAT miR-323linR6 22GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACAGAGGT miR-324-5linR6 23GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACACACCA let7aLinR6 24GTCGTATCCAGTGCAGGGTCCGAGGGTATTCGCACTGGATACGACAACTAT SEQ IDForward primer ID NO: Forward primer sequences miR-16F55 25CGCGCTAGCAGCACGTAAAT miR-20F56 26 GCCGCTAAAGTGCTTATAGTGC miR-21F56 27GCCCGCTAGCTTATCAGACTGATG miR-22F56 28 GCCTGAAGCTGCCAGTTGA miR-26aF54 29CCGGCGTTCAAGTAATCCAGGA miR-29F56 30 GCCGCTAGCACCATCTGAAA miR-30aF58 31GCCCCTTTCAGTCGGATGTTT miR-34F56 32 GCCCGTGGCAGTGTCTTAG miR-200bF56 33GCCCCTCTAATACTGCCTGG miR-323F58 34 GCCACGCACATTACACGGTC miR-324-5F56 35GCCACCATCCCCTAGGGC let-7a1F56 36 GCCGCTGAGGTAGTAGGTTGT SEQ IDTaqMan probe ID NO: TaqMan probe sequences miR-16_Tq8F67 37(6FAM)ATACGACCGCCAATAT(MGB) miR20_Tq8F68 38 (6FAM)CTGGATACGACTACCTG(MGB)miR-21_Tq8F68 39 (6FAM)CTGGATACGACTCAACA(MGB) miR-22_Tq8F68 40(6FAM)TGGATACGACACAGTTCT(MGB) miR-26a_Tq8F69 41(6FAM)TGGATACGACAGCCTATC(MGB) miR-29_Tq8F68 42(6FAM)TGGATACGACAACCGAT(MGB) miR30_Tq8F68 43 (6FAM)CTGGATACGACGCTGC(MGB)miR-34_Tq8F68 44 (6FAM)ATACGACACAACCAGC(MGB) miR-200b_Tq8F67 45(6FAM)ATACGACCATCATTACC(MGB) miR-323_Tq8F67 46(6FAM)CTGGATACGACAGAGGT(MGB) miR-324-5Tq8F68 47(6FAM)ATACGACACACCAATGC(MGB) let7a_Tq8F68 48(6FAM)TGGATACGACAACTATAC(MGB) SEQ ID Universal reverse primer ID NO:Reverse primer sequence miR-UP-R67.8 49 GTGCAGGGTCCGAGGT

Example 2

A multiplex (12-plex) assay was performed and the results compared to acorresponding collection of single-plex reactions. Additionally, theeffect of the presence or absence of ligase, as well as the presence orabsence of reverse transcriptase, was determined. The experiments wereperformed essentially the same as in Example 1, and the concentration ofeach linker in the 12-plex reaction was 0.05 uM, thereby resulting in atotal linker probe concentration of 0.6 uM. Further, the diluted 12-plexreverse transcription product was split into 12 different PCRamplification reactions, wherein a miRNA forward primer and a universalreverse primer and a detector probe where in each amplificationreaction. The miRNA sequences, Forward primers, and TaqMan detectorprobes are included in Table 2. The results are shown in Table 3.

TABLE 3 Singleplex vs. Multiplex Assay With Or Without T4 DNA Ligase1-plex Ct 12-plex Ct Ligation + RT 1-vs. miRNA Ligation + RT RT onlyLigation + RT RT only vs RT only 12-plex let-7a1 17.8 16.3 17.6 17.0 1.0−0.3 mir-16 16.0 15.1 16.1 15.3 0.9 −0.1 mir-20 19.3 18.7 19.8 19.5 0.4−0.6 mir-21 17.0 15.8 17.1 16.3 1.0 −0.3 mir-22 21.6 20.4 21.4 20.7 1.0−0.1 mir-26a 15.2 14.3 15.6 14.9 0.8 −0.4 mir-29 17.9 16.8 17.7 17.0 0.90.0 mir-30a 20.7 19.9 21.2 20.7 0.7 −0.7 mir-34 21.3 20.4 22.0 21.0 0.9−0.6 mir-200b 19.9 19.2 21.1 20.2 0.8 −1.0 mir-323 32.5 31.2 33.6 32.31.3 −1.1 mir-324-5 24.7 23.1 25.0 24.4 1.1 −0.8 Average 20.3 19.3 20.719.9 0.9 −0.5

Example 3

An experiment was performed to determine the effect of buffer conditionson reaction performance. In one set of experiments, a commerciallyavailable reverse transcription buffer from Applied Biosystems (partnumber 43400550) was employed in the hybridization and extensionreaction. In a corresponding set of experiments, a commerciallyavailable T4 DNA ligase buffer (NEB) was employed in the hybridizationand extension reaction. The experiments were performed as single-plexformat essentially as described for Example 1, and each miRNA was donein triplicate. The results are shown in Table 4, comparing RT buffer (ABpart #4340550) vs T4 DNA ligase buffer.

TABLE 4 T4 DNA Ligase RT vs RT Buffer Buffer T4 I II III Mean I II IIIMean Buffer let-7a1 22.7 22.8 22.8 22.8 20.8 20.7 20.6 20.7 2.1 mir-1618.4 18.5 18.6 18.5 17.7 17.8 17.9 17.8 0.7 mir-20 23.6 23.7 23.8 23.723.1 23.1 23.0 23.1 0.6 mir-21 20.4 20.4 20.5 20.4 19.4 19.3 19.2 19.31.1 mir-22 24.0 23.9 24.1 24.0 22.7 22.7 22.7 22.7 1.3 mir-26a 19.8 19.920.1 19.9 18.9 19.0 19.0 18.9 1.0 mir-29 21.3 21.3 21.4 21.3 20.5 20.620.5 20.5 0.8 mir-30a 24.4 24.4 24.4 24.4 23.6 23.4 23.6 23.5 0.9 mir-3424.9 24.8 25.1 25.0 23.0 23.1 23.2 23.1 1.9 mir- 25.8 25.8 25.9 25.924.6 24.6 24.8 24.7 1.2 200b mir-323 34.6 34.5 34.8 34.6 34.7 34.2 34.534.5 0.2 mir- 26.0 26.0 26.1 26.0 25.4 25.7 25.6 25.6 0.5 324-5 Average23.8 23.8 24.0 23.9 22.9 22.8 22.9 22.9 1.0

Example 4

An experiment was performed to examine the effect of ligase and kinasein a real-time miRNA amplification reaction. Here, twelve single-plexreactions were performed in duplicate, essentially as described inExample 1. Results are shown in Table 5.

TABLE 5 Ligase & Kinase No Ligase/No Kinase I II Mean I II Mean let-7a117.7 17.9 17.8 16.2 16.4 16.3 mir-16 15.9 16.2 16.0 15.0 15.2 15.1mir-20 19.1 19.6 19.3 18.6 18.9 18.7 mir-21 16.9 17.2 17.0 15.7 15.915.8 mir-22 21.4 21.7 21.6 20.3 20.5 20.4 mir-26a 15.0 15.4 15.2 14.314.4 14.3 mir-29 17.9 18.0 17.9 16.7 16.8 16.8 mir-30a 20.6 20.8 20.719.8 20.0 19.9 mir-34 21.1 21.5 21.3 20.4 20.5 20.4 mir-200b 19.8 20.019.9 19.2 19.3 19.2 mir-323 32.3 32.6 32.5 31.1 31.2 31.2 mir-324-5 24.624.8 24.7 23.0 23.3 23.1 Average 20.2 20.5 20.3 19.2 19.4 19.3

Example 5

An experiment was performed to determine the effect of sample materialon Ct values in a real-time miRNA amplification reaction. Here, cells,GuHCl lysate, Tris lysate, and Purified RNA were compared. The cellswere NIH3T3 cells. The Purified RNA was collected using the commerciallyavailable mirVana mRNA isolation kit for Ambion (catalog number 1560).ΔTris lysate, and a Guanidine lysate (GuHCl) (commercially availablefrom Applied Biosystems), were prepared as follows:

For the Tris lysate, a 1× lysis buffer comprised 10 mM Tris-HCl, pH 8.0,0.02% Sodium Azide, and 0.03% Tween-20. Trypsinized cells were pelletedby centrifugation at 1500 rpm for 5 minutes. The growth media wasremoved by aspiration, being careful that the cell pellet was notdisturbed. PBS was added to bring the cells to 2×10³ cells/ul. Next 10ul of cell suspension was mixed with 10 ul of a 2× lysis buffer and spunbriefly. The tubes were then immediately incubated for 5 minutes at 95C, and then immediately placed in a chilled block on ice for 2 minutes.The tubes were then mixed well and spun briefly at full speed before use(or optionally, stored at −20 C).

For the GuHCl lysate, a 1× lysis buffer comprised 2.5M GuHCl, 150 mM MESpH 6.0, 200 mM NaCl, 0.75% Tween-20. Trypsinized cells were pelleted bycentrifugation at 1500 rpm for 5 minutes. The growth media was removedby aspiration, being careful that the cell pellet was not disturbed. Thecell pellet was then re-suspended in 1×PBS, Ca++ and Mg++ free to bringcells to 2×10⁴ cells/uL. Then, 1 volume of 2× lysis buffer was added. Toensure complete nucleic acid release, this was followed by pipetting upand down ten times, followed by a brief spin. Results are shown in Table6.

Similar results were obtained for a variety of cell lines, includingNIH/3T3, OP9, A549, and HepG2 cells.

TABLE 6 Ct miRNA ID Cells GuHCl lysate Tris lysate Purified RNA let-7a124.9 31.3 28.2 31.5 mir-16 22.3 25.2 22.3 24.9 mir-20 22.7 26.0 24.126.1 mir-21 21.3 24.2 22.0 24.7 mir-22 30.3 28.6 27.2 28.8 mir-26a 25.631.0 27.9 31.4 mir-29 27.2 27.9 26.5 27.4 mir-30a 26.1 32.2 28.9 30.7mir-34 26.8 30.3 26.4 27.4 mir-200b 40.0 40.0 40.0 40.0 mir-323 30.134.7 31.1 31.8 mir-324-5 28.6 29.7 28.3 29.3 Average 27.2 30.1 27.8 29.5

Example 6

An experiment was performed to demonstrate the ability of the reactionto selectively quantity mature miRNA in the presence of precursor miRNA.Here, let-7a miRNA and mir-26b miRNA were queried in both mature form aswell as in their precursor form. Experiments were performed essentiallyas described for Example 1 in the no ligase condition, done intriplicate, with varying amounts of target material as indicated.Results are shown in Table 7. The sequences examined were as follows:

Mature let-7a, Seq ID NO: 50 UGAGGUAGUAGGUUGUAUAGUUPrecursor let-7a, SEQ ID NO: 51 (Note that theunderlined sequences corresponds to the Mature let-7a.)GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUCUGCCCUGCUAUGGGAUAACUAUACAAUCUACUGUCUUUCCU Mature mir-26b, SEQ ID NO: 52UUCAAGUAAUUCAGGAUAGGU Precursor mir-26b of SEQ ID NO: 53 (Note that theunderlined sequences corresponds to the Mature mir-26b.)CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUUGUGUGCUGUCCAGCCUGUUCUCCAUUACUUGGCUCGGGGACCGG

TABLE 7 Mouse Synthetic Synthetic lung miRNA precursor Assay specificfor (CT) Target RNA (ng) (fM) (fM) miRNA Precursor Let-7a 0 0 0 40.0 ±0.0 40.0 ± 0.0 (let-7a3) 0 10 0 24.2 ± 0.3 40.0 ± 0.0 0 100 0 21.0 ± 0.240.0 ± 0.0 0 0 10 35.0 ± 1.0 25.0 ± 0.1 0 0 100 31.0 ± 0.1 21.5 ± 0.1 100 0 19.1 ± 0.4 40.0 ± 0.0 Mir-26b 0 0 0 40.0 ± 0.0 40.0 ± 0.0 0 10 023.1 ± 0.1 40.0 ± 0.0 0 100 0 19.7 ± 0.1 40.0 ± 0.0 0 0 10 32.9 ± 0.425.7 ± 0.0 0 0 100 28.9 ± 0.2 22.3 ± 0.0 10 0 0 20.5 ± 0.1 28.0 ± 0.2

Example 7

An experiment was performed on synthetic let-7a miRNA to assess thenumber of 3′ nucleotides in the 3′ target specific portion of the linkerprobe that correspond with the 3′ end region of the miRNA. Theexperiment was performed as essentially as described supra for Example 1for the no ligase condition, and results are shown in Table 8 as meansand standard deviations of Ct values.

TABLE 8 miRNA assay components: let-7a miRNA synthetic target: let-7aNo. 3′ ssDNA linker probe target C_(T) values & statistics specificportion bases I II III Average SD 7 29.4 29.1 29.3 29.3 0.1 6 30.1 29.930.2 30.1 0.2 5 33.9 33.2 33.8 33.6 0.4 4 40.0 39.2 40.0 39.7 0.4In some embodiments, 3′ target specific portions of linker probespreferably comprise 5 nucleotides that correspond to the 3′ end regionof miRNAs. For example, miR-26a and miR-26b differ by only 2 bases, oneof which is the 3′ end nucleotide of miR-26a. Linker probes comprising 5nucleotides at their 3′ target specific portions can be employed toselectively detect miR-26a versus miR-26b.

Additional strategies for using the linker probes of the presentteachings in the context of single step assays, as well as in thecontext of short primer compositions, can be found in filed U.S.Provisional application “Compositions, Methods, and Kits for Identifyingand Quantitating Small RNA Molecules” by Lao and Straus, as well as inElfaitouri et al., J. Clin. Virol. 2004, 30(2): 150-156.

The present teachings further contemplate linker probe compositionscomprising 3′ target specific portions corresponding to any micro RNAsequence, including but without limitation, those sequences shown inTable 9, including C. elegans (cel), mouse (mmu), human (hsa),drosophila (dme), rat (rno), and rice (osa).

TABLE 9 SEQ ID NO: cel-let-7 54 ugagguaguagguuguauaguu cel-lin-4 55ucccugagaccucaaguguga cel-miR-1 56 uggaauguaaagaaguaugua cel-miR-2 57uaucacagccagcuuugaugugc cel-miR-34 58 aggcagugugguuagcugguug cel-miR-3559 ucaccggguggaaacuagcagu cel-miR-36 60 ucaccgggugaaaauucgcaugcel-miR-37 61 ucaccgggugaacacuugcagu cel-miR-38 62ucaccgggagaaaaacuggagu cel-miR-39 63 ucaccggguguaaaucagcuug cel-miR-4064 ucaccggguguacaucagcuaa cel-miR-41 65 ucaccgggugaaaaaucaccuacel-miR-42 66 caccggguuaacaucuacag cel-miR-43 67 uaucacaguuuacuugcugucgccel-miR-44 68 ugacuagagacacauucagcu cel-miR-45 69 ugacuagagacacauucagcucel-miR-46 70 ugucauggagucgcucucuuca cel-miR-47 71ugucauggaggcgcucucuuca cel-miR-48 72 ugagguaggcucaguagaugcga cel-miR-4973 aagcaccacgagaagcugcaga cel-miR-50 74 ugauaugucugguauucuuggguucel-miR-51 75 uacccguagcuccuauccauguu cel-miR-52 76cacccguacauauguuuccgugcu cel-miR-53 77 cacccguacauuuguuuccgugcucel-miR-54 78 uacccguaaucuucauaauccgag cel-miR-55 79uacccguauaaguuucugcugag cel-miR-56* 80 uggcggauccauuuuggguuguacel-miR-56 81 uacccguaauguuuccgcugag cel-miR-57 82uacccuguagaucgagcugugugu cel-miR-58 83 ugagaucguucaguacggcaau cel-miR-5984 ucgaaucguuuaucaggaugaug cel-miR-60 85 uauuaugcacauuuucuaguucacel-miR-61 86 ugacuagaaccguuacucaucuc cel-miR-62 87ugauauguaaucuagcuuacag cel-miR-63 88 uaugacacugaagcgaguuggaaa cel-miR-6489 uaugacacugaagcguuaccgaa cel-miR-65 90 uaugacacugaagcguaaccgaacel-miR-66 91 caugacacugauuagggauguga cel-miR-67 92ucacaaccuccuagaaagaguaga cel-miR-68 93 ucgaagacucaaaaguguaga cel-miR-6994 ucgaaaauuaaaaaguguaga cel-miR-70 95 uaauacgucguugguguuuccaucel-miR-71 96 ugaaagacauggguaguga cel-miR-72 97 aggcaagauguuggcauagccel-miR-73 98 uggcaagauguaggcaguucagu cel-miR-74 99uggcaagaaauggcagucuaca cel-miR-75 100 uuaaagcuaccaaccggcuuca cel-miR-76101 uucguuguugaugaagccuuga cel-miR-77 102 uucaucaggccauagcuguccacel-miR-78 103 uggaggccugguuguuugugc cel-miR-79 104auaaagcuagguuaccaaagcu cel-miR-227 105 agcuuucgacaugauucugaac cel-miR-80106 ugagaucauuaguugaaagccga cel-miR-81 107 ugagaucaucgugaaagcuagucel-miR-82 108 ugagaucaucgugaaagccagu cel-miR-83 109uagcaccauauaaauucaguaa cel-miR-84 110 ugagguaguauguaauauugua cel-miR-85111 uacaaaguauuugaaaagucgugc cel-miR-86 112 uaagugaaugcuuugccacaguccel-miR-87 113 gugagcaaaguuucaggugu cel-miR-90 114ugauauguuguuugaaugcccc cel-miR-124 115 uaaggcacgcggugaaugcca cel-miR-228116 aauggcacugcaugaauucacgg cel-miR-229 117 aaugacacugguuaucuuuuccaucgucel-miR-230 118 guauuaguugugcgaccaggaga cel-miR-231 119uaagcucgugaucaacaggcagaa cel-miR-232 120 uaaaugcaucuuaacugcggugacel-miR-233 121 uugagcaaugcgcaugugcggga cel-miR-234 122uuauugcucgagaauacccuu cel-miR-235 123 uauugcacucuccccggccuga cel-miR-236124 uaauacugucagguaaugacgcu cel-miR-237 125 ucccugagaauucucgaacagcuucel-miR-238 126 uuuguacuccgaugccauucaga cel-miR-239a 127uuuguacuacacauagguacugg cel-miR-239b 128 uuguacuacacaaaaguacugcel-miR-240 129 uacuggcccccaaaucuucgcu cel-miR-241 130ugagguaggugcgagaaauga cel-miR-242 131 uugcguaggccuuugcuucga cel-miR-243132 cgguacgaucgcggcgggauauc cel-miR-244 133 ucuuugguuguacaaagugguaugcel-miR-245 134 auugguccccuccaaguagcuc cel-miR-246 135uuacauguuucggguaggagcu cel-miR-247 136 ugacuagagccuauucucuucuucel-miR-248 137 uacacgugcacggauaacgcuca cel-miR-249 138ucacaggacuuuugagcguugc cel-miR-250 139 ucacagucaacuguuggcauggcel-miR-251 140 uuaaguaguggugccgcucuuauu cel-miR-252 141uaaguaguagugccgcagguaac cel-miR-253 142 cacaccucacuaacacugacccel-miR-254 143 ugcaaaucuuucgcgacuguagg cel-miR-256 144uggaaugcauagaagacugua cel-miR-257 145 gaguaucaggaguacccaguga cel-miR-258146 gguuuugagaggaauccuuuu cel-miR-259 147 aaaucucauccuaaucugguacel-miR-260 148 gugaugucgaacucuuguag cel-miR-261 149 uagcuuuuuaguuuucacgcel-miR-262 150 guuucucgauguuuucugau cel-miR-264 151ggcgggugguuguuguuaug cel-miR-265 152 ugagggaggaagggugguau cel-miR-266153 aggcaagacuuuggcaaagc cel-miR-267 154 cccgugaagugucugcugcacel-miR-268 155 ggcaagaauuagaagcaguuuggu cel-miR-269 156ggcaagacucuggcaaaacu cel-miR-270 157 ggcaugauguagcaguggag cel-miR-271158 ucgccgggugggaaagcauu cel-miR-272 159 uguaggcauggguguuug cel-miR-273160 ugcccguacugugucggcug cel-miR-353 161 caauugccauguguugguauucel-miR-354 162 accuuguuuguugcugcuccu cel-miR-355 163uuuguuuuagccugagcuaug cel-miR-356 164 uugagcaacgcgaacaaauca cel-miR-357165 uaaaugccagucguugcagga cel-miR-358 166 caauugguaucccugucaaggcel-miR-359 167 ucacuggucuuucucugacga cel-miR-360 168ugaccguaaucccguucacaa cel-lsy-6 169 uuuuguaugagacgcauuucg cel-miR-392170 uaucaucgaucacgugugauga hsa-let-7a 171 ugagguaguagguuguauaguuhsa-let-7b 172 ugagguaguagguugugugguu hsa-let-7c 173ugagguaguagguuguaugguu hsa-let-7d 174 agagguaguagguugcauagu hsa-let-7e175 ugagguaggagguuguauagu hsa-let-7f 176 ugagguaguagauuguauaguuhsa-miR-15a 177 uagcagcacauaaugguuugug hsa-miR-16 178uagcagcacguaaauauuggcg hsa-miR-17-5p 179 caaagugcuuacagugcagguaguhsa-miR-17-3p 180 acugcagugaaggcacuugu hsa-miR-18 181uaaggugcaucuagugcagaua hsa-miR-19a 182 ugugcaaaucuaugcaaaacugahsa-miR-19b 183 ugugcaaauccaugcaaaacuga hsa-miR-20 184uaaagugcuuauagugcaggua hsa-miR-21 185 uagcuuaucagacugauguuga hsa-miR-22186 aagcugccaguugaagaacugu hsa-miR-23a 187 aucacauugccagggauuucchsa-miR-189 188 gugccuacugagcugauaucagu hsa-miR-24 189uggcucaguucagcaggaacag hsa-miR-25 190 cauugcacuugucucggucuga hsa-miR-26a191 uucaaguaauccaggauaggcu hsa-miR-26b 192 uucaaguaauucaggauagguhsa-miR-27a 193 uucacaguggcuaaguuccgcc hsa-miR-28 194aaggagcucacagucuauugag hsa-miR-29a 195 cuagcaccaucugaaaucgguuhsa-miR-30a* 196 uguaaacauccucgacuggaagc hsa-miR-30a 197cuuucagucggauguuugcagc hsa-miR-31 198 ggcaagaugcuggcauagcug hsa-miR-32199 uauugcacauuacuaaguugc hsa-miR-33 200 gugcauuguaguugcauug hsa-miR-92201 uauugcacuugucccggccugu hsa-miR-93 202 aaagugcuguucgugcagguaghsa-miR-95 203 uucaacggguauuuauugagca hsa-miR-96 204uuuggcacuagcacauuuuugc hsa-miR-98 205 ugagguaguaaguuguauuguu hsa-miR-99a206 aacccguagauccgaucuugug hsa-miR-100 207 aacccguagauccgaacuugughsa-miR-101 208 uacaguacugugauaacugaag hsa-miR-29b 209uagcaccauuugaaaucagu hsa-miR-103 210 agcagcauuguacagggcuauga hsa-miR-105211 ucaaaugcucagacuccugu hsa-miR-106a 212 aaaagugcuuacagugcagguagchsa-miR-107 213 agcagcauuguacagggcuauca hsa-miR-192 214cugaccuaugaauugacagcc hsa-miR-196 215 uagguaguuucauguuguugg hsa-miR-197216 uucaccaccuucuccacccagc hsa-miR-198 217 gguccagaggggagauagghsa-miR-199a 218 cccaguguucagacuaccuguuc hsa-miR-199a* 219uacaguagucugcacauugguu hsa-miR-208 220 auaagacgagcaaaaagcuuguhsa-miR-148a 221 ucagugcacuacagaacuuugu hsa-miR-30c 222uguaaacauccuacacucucagc hsa-miR-30d 223 uguaaacauccccgacuggaaghsa-miR-139 224 ucuacagugcacgugucu hsa-miR-147 225 guguguggaaaugcuucugchsa-miR-7 226 uggaagacuagugauuuuguu hsa-miR-10a 227uacccuguagauccgaauuugug hsa-miR-10b 228 uacccuguagaaccgaauuuguhsa-miR-34a 229 uggcagugucuuagcugguugu hsa-miR-181a 230aacauucaacgcugucggugagu hsa-miR-181b 231 aacauucauugcugucgguggguuhsa-miR-181c 232 aacauucaaccugucggugagu hsa-miR-182 233uuuggcaaugguagaacucaca hsa-miR-182* 234 ugguucuagacuugccaacuahsa-miR-183 235 uauggcacugguagaauucacug hsa-miR-187 236ucgugucuuguguugcagccg hsa-miR-199b 237 cccaguguuuagacuaucuguuchsa-miR-203 238 gugaaauguuuaggaccacuag hsa-miR-204 239uucccuuugucauccuaugccu hsa-miR-205 240 uccuucauuccaccggagucughsa-miR-210 241 cugugcgugugacagcggcug hsa-miR-211 242uucccuuugucauccuucgccu hsa-miR-212 243 uaacagucuccagucacggcc hsa-miR-213244 accaucgaccguugauuguacc hsa-miR-214 245 acagcaggcacagacaggcaghsa-miR-215 246 augaccuaugaauugacagac hsa-miR-216 247uaaucucagcuggcaacugug hsa-miR-217 248 uacugcaucaggaacugauuggauhsa-miR-218 249 uugugcuugaucuaaccaugu hsa-miR-219 250ugauuguccaaacgcaauucu hsa-miR-220 251 ccacaccguaucugacacuuu hsa-miR-221252 agcuacauugucugcuggguuuc hsa-miR-222 253 agcuacaucuggcuacugggucuchsa-miR-223 254 ugucaguuugucaaauacccc hsa-miR-224 255caagucacuagugguuccguuua hsa-miR-200b 256 cucuaauacugccugguaaugaughsa-let-7g 257 ugagguaguaguuuguacagu hsa-let-7i 258 ugagguaguaguuugugcuhsa-miR-1 259 uggaauguaaagaaguaugua hsa-miR-15b 260uagcagcacaucaugguuuaca hsa-miR-23b 261 aucacauugccagggauuaccachsa-miR-27b 262 uucacaguggcuaaguucug hsa-miR-30b 263uguaaacauccuacacucagc hsa-miR-122a 264 uggagugugacaaugguguuuguhsa-miR-124a 265 uuaaggcacgcggugaaugcca hsa-miR-125b 266ucccugagacccuaacuuguga hsa-miR-128a 267 ucacagugaaccggucucuuuuhsa-miR-130a 268 cagugcaauguuaaaagggc hsa-miR-132 269uaacagucuacagccauggucg hsa-miR-133a 270 uugguccccuucaaccagcuguhsa-miR-135a 271 uauggcuuuuuauuccuauguga hsa-miR-137 272uauugcuuaagaauacgcguag hsa-miR-138 273 agcugguguugugaauc hsa-miR-140 274agugguuuuacccuaugguag hsa-miR-141 275 aacacugucugguaaagaugghsa-miR-142-5p 276 cauaaaguagaaagcacuac hsa-miR-142-3p 277uguaguguuuccuacuuuaugga hsa-miR-143 278 ugagaugaagcacuguagcucahsa-miR-144 279 uacaguauagaugauguacuag hsa-miR-145 280guccaguuuucccaggaaucccuu hsa-miR-152 281 ucagugcaugacagaacuugghsa-miR-153 282 uugcauagucacaaaaguga hsa-miR-191 283caacggaaucccaaaagcagcu hsa-miR-9 284 ucuuugguuaucuagcuguauga hsa-miR-9*285 uaaagcuagauaaccgaaagu hsa-miR-125a 286 ucccugagacccuuuaaccugughsa-miR-126* 287 cauuauuacuuuugguacgcg hsa-miR-126 288ucguaccgugaguaauaaugc hsa-miR-127 289 ucggauccgucugagcuuggcu hsa-miR-129290 cuuuuugcggucugggcuugc hsa-miR-134 291 ugugacugguugaccagaggghsa-miR-136 292 acuccauuuguuuugaugaugga hsa-miR-146 293ugagaacugaauuccauggguu hsa-miR-149 294 ucuggcuccgugucuucacucchsa-miR-150 295 ucucccaacccuuguaccagug hsa-miR-154 296uagguuauccguguugccuucg hsa-miR-184 297 uggacggagaacugauaaggguhsa-miR-185 298 uggagagaaaggcaguuc hsa-miR-186 299caaagaauucuccuuuugggcuu hsa-miR-188 300 caucccuugcaugguggaggguhsa-miR-190 301 ugauauguuugauauauuaggu hsa-miR-193 302aacuggccuacaaagucccag hsa-miR-194 303 uguaacagcaacuccaugugga hsa-miR-195304 uagcagcacagaaauauuggc hsa-miR-206 305 uggaauguaaggaagugugugghsa-miR-320 306 aaaagcuggguugagagggcgaa hsa-miR-321 307uaagccagggauuguggguuc hsa-miR-200c 308 aauacugccggguaaugauggahsa-miR-155 309 uuaaugcuaaucgugauagggg hsa-miR-128b 310ucacagugaaccggucucuuuc hsa-miR-106b 311 uaaagugcugacagugcagauhsa-miR-29c 312 uagcaccauuugaaaucgguua hsa-miR-200a 313uaacacugucugguaacgaugu hsa-miR-302 314 uaagugcuuccauguuuuggugahsa-miR-34b 315 aggcagugucauuagcugauug hsa-miR-34c 316aggcaguguaguuagcugauug hsa-miR-299 317 ugguuuaccgucccacauacauhsa-miR-301 318 cagugcaauaguauugucaaagc hsa-miR-99b 319cacccg uagaaccgaccuugcg hsa-miR-296 320 agggcccccccucaauccuguhsa-miR-130b 321 cagugcaaugaugaaagggcau hsa-miR-30e 322uguaaacauccuugacugga hsa-miR-340 323 uccgucucaguuacuuuauagcc hsa-miR-330324 gcaaagcacacggccugcagaga hsa-miR-328 325 cuggcccucucugcccuuccguhsa-miR-342 326 ucucacacagaaaucgcacccguc hsa-miR-337 327uccagcuccuauaugaugccuuu hsa-miR-323 328 gcacauuacacggucgaccucuhsa-miR-326 329 ccucugggcccuuccuccag hsa-miR-151 330acuagacugaagcuccuugagg hsa-miR-135b 331 uauggcuuuucauuccuaugughsa-miR-148b 332 ucagugcaucacagaacuuugu hsa-miR-331 333gccccugggccuauccuagaa hsa-miR-324-5p 334 cgcauccccuagggcauugguguhsa-miR-324-3p 335 ccacugccccaggugcugcugg hsa-miR-338 336uccagcaucagugauuuuguuga hsa-miR-339 337 ucccuguccuccaggagcucahsa-miR-335 338 ucaagagcaauaacgaaaaaugu hsa-miR-133b 339uugguccccuucaaccagcua osa-miR156 340 ugacagaagagagugagcac osa-miR160 341ugccuggcucccuguaugcca osa-miR162 342 ucgauaaaccucugcauccag osa-miR164343 uggagaagcagggcacgugca osa-miR166 344 ucggaccaggcuucauuccccosa-miR167 345 ugaagcugccagcaugaucua osa-miR169 346cagccaaggaugacuugccga osa-miR171 347 ugauugagccgcgccaauauc mmu-let-7g348 ugagguaguaguuuguacagu mmu-let-7i 349 ugagguaguaguuugugcu mmu-miR-1350 uggaauguaaagaaguaugua mmu-miR-15b 351 uagcagcacaucaugguuuacammu-miR-23b 352 aucacauugccagggauuaccac mmu-miR-27b 353uucacaguggcuaaguucug mmu-miR-29b 354 uagcaccauuugaaaucagugu mmu-miR-30a*355 uguaaacauccucgacuggaagc mmu-miR-30a 356 cuuucagucggauguuugcagcmmu-miR-30b 357 uguaaacauccuacacucagc mmu-miR-99a 358acccguagauccgaucuugu mmu-miR-99b 359 cacccguagaaccgaccuugcg mmu-miR-101360 uacaguacugugauaacuga mmu-miR-124a 361 uuaaggcacgcggugaaugccammu-miR-125a 362 ucccugagacccuuuaaccugug mmu-miR-125b 363ucccugagacccuaacuuguga mmu-miR-126* 364 cauuauuacuuuugguacgcgmmu-miR-126 365 ucguaccgugaguaauaaugc mmu-miR-127 366ucggauccgucugagcuuggcu mmu-miR-128a 367 ucacagugaaccggucucuuuummu-miR-130a 368 cagugcaauguuaaaagggc mmu-miR-9 369ucuuugguuaucuagcuguauga mmu-miR-9* 370 uaaagcuagauaaccgaaagu mmu-miR-132371 uaacagucuacagccauggucg mmu-miR-133a 372 uugguccccuucaaccagcugummu-miR-134 373 ugugacugguugaccagaggg mmu-miR-135a 374uauggcuuuuuauuccuauguga mmu-miR-136 375 acuccauuuguuuugaugauggammu-miR-137 376 uauugcuuaagaauacgcguag mmu-miR-138 377 agcugguguugugaaucmmu-miR-140 378 agugguuuuacccuaugguag mmu-miR-141 379aacacugucugguaaagaugg mmu-miR-142-5p 380 cauaaaguagaaagcacuacmmu-miR-142-3p 381 uguaguguuuccuacuuuaugg mmu-miR-144 382uacaguauagaugauguacuag mmu-miR-145 383 guccaguuuucccaggaaucccuummu-miR-146 384 ugagaacugaauuccauggguu mmu-miR-149 385ucuggcuccgugucuucacucc mmu-miR-150 386 ucucccaacccuuguaccagugmmu-miR-151 387 cuagacugaggcuccuugagg mmu-miR-152 388ucagugcaugacagaacuugg mmu-miR-153 389 uugcauagucacaaaaguga mmu-miR-154390 uagguuauccguguugccuucg mmu-miR-155 391 uuaaugcuaauugugauaggggmmu-miR-10b 392 cccuguagaaccgaauuugugu mmu-miR-129 393cuuuuugcggucugggcuugcu mmu-miR-181a 394 aacauucaacgcugucggugagummu-miR-182 395 uuuggcaaugguagaacucaca mmu-miR-183 396uauggcacugguagaauucacug mmu-miR-184 397 uggacggagaacugauaagggummu-miR-185 398 uggagagaaaggcaguuc mmu-miR-186 399caaagaauucuccuuuugggcuu mmu-miR-187 400 ucgugucuuguguugcagccggmmu-miR-188 401 caucccuugcaugguggagggu mmu-miR-189 402gugccuacugagcugauaucagu mmu-miR-24 403 uggcucaguucagcaggaacagmmu-miR-190 404 ugauauguuugauauauuaggu mmu-miR-191 405caacggaaucccaaaagcagcu mmu-miR-193 406 aacuggccuacaaagucccag mmu-miR-194407 uguaacagcaacuccaugugga mmu-miR-195 408 uagcagcacagaaauauuggcmmu-miR-199a 409 cccaguguucagacuaccuguuc mmu-miR-199a* 410uacaguagucugcacauugguu mmu-miR-200b 411 uaauacugccugguaaugaugacmmu-miR-201 412 uacucaguaaggcauuguucu mmu-miR-202 413agagguauagcgcaugggaaga mmu-miR-203 414 ugaaauguuuaggaccacuag mmu-miR-204415 uucccuuugucauccuaugccug mmu-miR-205 416 uccuucauuccaccggagucugmmu-miR-206 417 uggaauguaaggaagugugugg mmu-miR-207 418gcuucuccuggcucuccucccuc mmu-miR-122a 419 uggagugugacaaugguguuugummu-miR-143 420 ugagaugaagcacuguagcuca mmu-miR-30e 421uguaaacauccuugacugga mmu-miR-290 422 cucaaacuaugggggcacuuuuummu-miR-291-5p 423 caucaaaguggaggcccucucu mmu-miR-291-3p 424aaagugcuuccacuuugugugcc mmu-miR-292-5p 425 acucaaacugggggcucuuuugmmu-miR-292-3p 426 aagugccgccagguuuugagugu mmu-miR-293 427agugccgcagaguuuguagugu mmu-miR-294 428 aaagugcuucccuuuugugugummu-miR-295 429 aaagugcuacuacuuuugagucu mmu-miR-296 430agggcccccccucaauccugu mmu-miR-297 431 auguaugugugcaugugcaug mmu-miR-298432 ggcagaggagggcuguucuucc mmu-miR-299 433 ugguuuaccgucccacauacaummu-miR-300 434 uaugcaagggcaagcucucuuc mmu-miR-301 435cagugcaauaguauugucaaagc mmu-miR-302 436 uaagugcuuccauguuuuggugammu-miR-34c 437 aggcaguguaguuagcugauugc mmu-miR-34b 438uaggcaguguaauuagcugauug mmu-let-7d 439 agagguaguagguugcauagu mmu-let-7d*440 cuauacgaccugcugccuuucu mmu-miR-106a 441 caaagugcuaacagugcagguammu-miR-106b 442 uaaagugcugacagugcagau mmu-miR-130b 443cagugcaaugaugaaagggcau mmu-miR-19b 444 ugugcaaauccaugcaaaacugammu-miR-30c 445 uguaaacauccuacacucucagc mmu-miR-30d 446uguaaacauccccgacuggaag mmu-miR-148a 447 ucagugcacuacagaacuuugummu-miR-192 448 cugaccuaugaauugaca mmu-miR-196 449 uagguaguuucauguuguuggmmu-miR-200a 450 uaacacugucugguaacgaugu mmu-miR-208 451auaagacgagcaaaaagcuugu mmu-let-7a 452 ugagguaguagguuguauaguu mmu-let-7b453 ugagguaguagguugugugguu mmu-let-7c 454 ugagguaguagguuguaugguummu-let-7e 455 ugagguaggagguuguauagu mmu-let-7f 456ugagguaguagauuguauaguu mmu-miR-15a 457 uagcagcacauaaugguuugug mmu-miR-16458 uagcagcacguaaauauuggcg mmu-miR-18 459 uaaggugcaucuagugcagauammu-miR-20 460 uaaagugcuuauagugcagguag mmu-miR-21 461uagcuuaucagacugauguuga mmu-miR-22 462 aagcugccaguugaagaacugu mmu-miR-23a463 aucacauugccagggauuucc mmu-miR-26a 464 uucaaguaauccaggauaggcummu-miR-26b 465 uucaaguaauucaggauagguu mmu-miR-29a 466cuagcaccaucugaaaucgguu mmu-miR-29c 467 uagcaccauuugaaaucgguuammu-miR-27a 468 uucacaguggcuaaguuccgc mmu-miR-31 469aggcaagaugcuggcauagcug mmu-miR-92 470 uauugcacuugucccggccug mmu-miR-93471 caaagugcuguucgugcagguag mmu-miR-96 472 uuuggcacuagcacauuuuugcummu-miR-34a 473 uggcagugucuuagcugguuguu mmu-miR-98 474ugagguaguaaguuguauuguu mmu-miR-103 475 agcagcauuguacagggcuaugammu-miR-323 476 gcacauuacacggucgaccucu mmu-miR-324-5p 477cgcauccccuagggcauuggugu mmu-miR-324-3p 478 ccacugccccaggugcugcuggmmu-miR-325 479 ccuaguaggugcucaguaagugu mmu-miR-326 480ccucugggcccuuccuccagu mmu-miR-328 481 cuggcccucucugcccuuccgu mmu-miR-329482 aacacacccagcuaaccuuuuu mmu-miR-330 483 gcaaagcacagggccugcagagammu-miR-331 484 gccccugggccuauccuagaa mmu-miR-337 485uucagcuccuauaugaugccuuu mmu-miR-338 486 uccagcaucagugauuuuguugammu-miR-339 487 ucccuguccuccaggagcuca mmu-miR-340 488uccgucucaguuacuuuauagcc mmu-miR-341 489 ucgaucggucggucggucagummu-miR-342 490 ucucacacagaaaucgcacccguc mmu-miR-344 491ugaucuagccaaagccugacugu mmu-miR-345 492 ugcugaccccuaguccagugcmmu-miR-346 493 ugucugcccgagugccugccucu mmu-miR-350 494uucacaaagcccauacacuuucac mmu-miR-135b 495 uauggcuuuucauuccuaugugmmu-miR-101b 496 uacaguacugugauagcugaag mmu-miR-107 497agcagcauuguacagggcuauca mmu-miR-10a 498 uacccuguagauccgaauuugugmmu-miR-17-5p 499 caaagugcuuacagugcagguagu mmu-miR-17-3p 500acugcagugagggcacuugu mmu-miR-19a 501 ugugcaaaucuaugcaaaacuga mmu-miR-25502 cauugcacuugucucggucuga mmu-miR-28 503 aaggagcucacagucuauugagmmu-miR-32 504 uauugcacauuacuaaguugc mmu-miR-100 505aacccguagauccgaacuugug mmu-miR-139 506 ucuacagugcacgugucu mmu-miR-200c507 aauacugccggguaaugaugga mmu-miR-210 508 cugugcgugugacagcggcugmmu-miR-212 509 uaacagucuccagucacggcc mmu-miR-213 510accaucgaccguugauuguacc mmu-miR-214 511 acagcaggcacagacaggcag mmu-miR-216512 uaaucucagcuggcaacugug mmu-miR-218 513 uugugcuugaucuaaccaugummu-miR-219 514 ugauuguccaaacgcaauucu mmu-miR-223 515ugucaguuugucaaauacccc mmu-miR-320 516 aaaagcuggguugagagggcgaammu-miR-321 517 uaagccagggauuguggguuc mmu-miR-33 518 gugcauuguaguugcauugmmu-miR-211 519 uucccuuugucauccuuugccu mmu-miR-221 520agcuacauugucugcuggguuu mmu-miR-222 521 agcuacaucuggcuacugggucummu-miR-224 522 uaagucacuagugguuccguuua mmu-miR-199b 523cccaguguuuagacuaccuguuc mmu-miR-181b 524 aacauucauugcugucgguggguummu-miR-181c 525 aacauucaaccugucggugagu mmu-miR-128b 526ucacagugaaccggucucuuuc mmu-miR-7 527 uggaagacuagugauuuuguu mmu-miR-7b528 uggaagacuugugauuuuguu mmu-miR-217 529 uacugcaucaggaacugacuggaummu-miR-133b 530 uugguccccuucaaccagcua mmu-miR-215 531augaccuaugauuugacagac dme-miR-1 532 uggaauguaaagaaguauggag dme-miR-2a533 uaucacagccagcuuugaugagc dme-miR-2b 534 uaucacagccagcuuugaggagcdme-miR-3 535 ucacugggcaaagugugucuca dme-miR-4 536 auaaagcuagacaaccauugadme-miR-5 537 aaaggaacgaucguugugauaug dme-miR-6 538uaucacaguggcuguucuuuuu dme-miR-7 539 uggaagacuagugauuuuguugu dme-miR-8540 uaauacugucagguaaagauguc dme-miR-9a 541 ucuuugguuaucuagcuguaugadme-miR-10 542 acccuguagauccgaauuugu dme-miR-11 543caucacagucugaguucuugc dme-miR-12 544 ugaguauuacaucagguacuggu dme-miR-13a545 uaucacagccauuuugaugagu dme-miR-13b 546 uaucacagccauuuugacgagudme-miR-14 547 ucagucuuuuucucucuccua dme-miR-263a 548guuaauggcacuggaagaauucac dme-miR-184* 549 ccuuaucauucucucgccccgdme-miR-184 550 uggacggagaacugauaagggc dme-miR-274 551uuuugugaccgacacuaacggguaau dme-miR-275 552 ucagguaccugaaguagcgcgcgdme-miR-92a 553 cauugcacuugucccggccuau dme-miR-219 554ugauuguccaaacgcaauucuug dme-miR-276a* 555 cagcgagguauagaguuccuacgdme-miR-276a 556 uaggaacuucauaccgugcucu dme-miR-277 557uaaaugcacuaucugguacgaca dme-miR-278 558 ucggugggacuuucguccguuudme-miR-133 559 uugguccccuucaaccagcugu dme-miR-279 560ugacuagauccacacucauuaa dme-miR-33 561 aggugcauuguagucgcauug dme-miR-280562 uguauuuacguugcauaugaaaugaua dme-miR-281-1* 563aagagagcuguccgucgacagu dme-miR-281 564 ugucauggaauugcucucuuugudme-miR-282 565 aaucuagccucuacuaggcuuugucugu dme-miR-283 566uaaauaucagcugguaauucu dme-miR-284 567 ugaagucagcaacuugauuccagcaauugdme-miR-281-2* 568 aagagagcuauccgucgacagu dme-miR-34 569uggcagugugguuagcugguug dme-miR-124 570 uaaggcacgcggugaaugccaagdme-miR-79 571 uaaagcuagauuaccaaagcau dme-miR-276b* 572cagcgagguauagaguuccuacg dme-miR-276b 573 uaggaacuuaauaccgugcucudme-miR-210 574 uugugcgugugacagcggcua dme-miR-285 575uagcaccauucgaaaucagugc dme-miR-100 576 aacccguaaauccgaacuugugdme-miR-92b 577 aauugcacuagucccggccugc dme-miR-286 578ugacuagaccgaacacucgugcu dme-miR-287 579 uguguugaaaaucguuugcac dme-miR-87580 uugagcaaaauuucaggugug dme-miR-263b 581 cuuggcacugggagaauucacdme-miR-288 582 uuucaugucgauuucauuucaug dme-miR-289 583uaaauauuuaaguggagccugcgacu dme-bantam 584 ugagaucauuuugaaagcugauudme-miR-303 585 uuuagguuucacaggaaacuggu dme-miR-31b 586uggcaagaugucggaauagcug dme-miR-304 587 uaaucucaauuuguaaaugugagdme-miR-305 588 auuguacuucaucaggugcucug dme-miR-9c 589ucuuugguauucuagcuguaga dme-miR-306 590 ucagguacuuagugacucucaadme-miR-306* 591 gggggucacucugugccugugc dme-miR-9b 592ucuuuggugauuuuagcuguaug dme-let-7 593 ugagguaguagguuguauagu dme-miR-125594 ucccugagacccuaacuuguga dme-miR-307 595 ucacaaccuccuugagugagdme-miR-308 596 aaucacaggauuauacugugag dme-miR-31a 597uggcaagaugucggcauagcuga dme-miR-309 598 gcacuggguaaaguuuguccuadme-miR-310 599 uauugcacacuucccggccuuu dme-miR-311 600uauugcacauucaccggccuga dme-miR-312 601 uauugcacuugagacggccugadme-miR-313 602 uauugcacuuuucacagcccga dme-miR-314 603uauucgagccaauaaguucgg dme-miR-315 604 uuuugauuguugcucagaaagc dme-miR-316605 ugucuuuuuccgcuuacuggcg dme-miR-317 606 ugaacacagcuggugguauccagudme-miR-318 607 ucacugggcuuuguuuaucuca dme-miR-2c 608uaucacagccagcuuugaugggc dme-miR-iab-4-5p 609 acguauacugaauguauccugadme-miR-iab-4-3p 610 cgguauaccuucaguauacguaac rno-miR-322 611aaacaugaagcgcugcaaca rno-miR-323 612 gcacauuacacggucgaccucu rno-miR-301613 cagugcaauaguauugucaaagcau rno-miR-324-5p 614 cgcauccccuagggcauuggugurno-miR-324-3p 615 ccacugccccaggugcugcugg rno-miR-325 616ccuaguaggugcucaguaagugu rno-miR-326 617 ccucugggcccuuccuccagu rno-let-7d618 agagguaguagguugcauagu rno-let-7d* 619 cuauacgaccugcugccuuucurno-miR-328 620 cuggcccucucugcccuuccgu rno-miR-329 621aacacacccagcuaaccuuuuu rno-miR-330 622 gcaaagcacagggccugcagagarno-miR-331 623 gccccugggccuauccuagaa rno-miR-333 624guggugugcuaguuacuuuu rno-miR-140 625 agugguuuuacccuaugguag rno-miR-140*626 uaccacaggguagaaccacggaca rno-miR-336 627 ucacccuuccauaucuagucurno-miR-337 628 uucagcuccuauaugaugccuuu rno-miR-148b 629ucagugcaucacagaacuuugu rno-miR-338 630 uccagcaucagugauuuuguugarno-miR-339 631 ucccuguccuccaggagcuca rno-miR-341 632ucgaucggucggucggucagu rno-miR-342 633 ucucacacagaaaucgcacccgucrno-miR-344 634 ugaucuagccaaagccugaccgu rno-miR-345 635ugcugaccccuaguccagugc rno-miR-346 636 ugucugccugagugccugccucurno-miR-349 637 cagcccugcugucuuaaccucu rno-miR-129 638cuuuuugcggucugggcuugcu rno-miR-129* 639 aagcccuuaccccaaaaagcaurno-miR-20 640 uaaagugcuuauagugcagguag rno-miR-20* 641acugcauuacgagcacuuaca rno-miR-350 642 uucacaaagcccauacacuuucac rno-miR-7643 uggaagacuagugauuuuguu rno-miR-7* 644 caacaaaucacagucugccauarno-miR-351 645 ucccugaggagcccuuugagccug rno-miR-135b 646uauggcuuuucauuccuaugug rno-miR-151* 647 ucgaggagcucacagucuaguarno-miR-151 648 acuagacugaggcuccuugagg rno-miR-101b 649uacaguacugugauagcugaag rno-let-7a 650 ugagguaguagguuguauaguu rno-let-7b651 ugagguaguagguugugugguu rno-let-7c 652 ugagguaguagguuguaugguurno-let-7e 653 ugagguaggagguuguauagu rno-let-7f 654ugagguaguagauuguauaguu rno-let-7i 655 ugagguaguaguuugugcu rno-miR-7b 656uggaagacuugugauuuuguu rno-miR-9 657 ucuuugguuaucuagcuguauga rno-miR-10a658 uacccuguagauccgaauuugug rno-miR-10b 659 uacccuguagaaccgaauuugurno-miR-15b 660 uagcagcacaucaugguuuaca rno-miR-16 661uagcagcacguaaauauuggcg rno-miR-17 662 caaagugcuuacagugcagguagurno-miR-18 663 uaaggugcaucuagugcagaua rno-miR-19b 664ugugcaaauccaugcaaaacuga rno-miR-19a 665 ugugcaaaucuaugcaaaacugarno-miR-21 666 uagcuuaucagacugauguuga rno-miR-22 667aagcugccaguugaagaacugu rno-miR-23a 668 aucacauugccagggauuucc rno-miR-23b669 aucacauugccagggauuaccac rno-miR-24 670 uggcucaguucagcaggaacagrno-miR-25 671 cauugcacuugucucggucuga rno-miR-26a 672uucaaguaauccaggauaggcu rno-miR-26b 673 uucaaguaauucaggauagguurno-miR-27b 674 uucacaguggcuaaguucug rno-miR-27a 675uucacaguggcuaaguuccgc rno-miR-28 676 aaggagcucacagucuauugag rno-miR-29b677 uagcaccauuugaaaucagugu rno-miR-29a 678 cuagcaccaucugaaaucgguurno-miR-29c 679 uagcaccauuugaaaucgguua rno-miR-30c 680uguaaacauccuacacucucagc rno-miR-30e 681 uguaaacauccuugacugga rno-miR-30b682 uguaaacauccuacacucagc rno-miR-30d 683 uguaaacauccccgacuggaagrno-miR-30a 684 cuuucagucggauguuugcagc rno-miR-31 685aggcaagaugcuggcauagcug rno-miR-32 686 uauugcacauuacuaaguugc rno-miR-33687 gugcauuguaguugcauug rno-miR-34b 688 uaggcaguguaauuagcugauugrno-miR-34c 689 aggcaguguaguuagcugauugc rno-miR-34a 690uggcagugucuuagcugguuguu rno-miR-92 691 uauugcacuugucccggccug rno-miR-93692 caaagugcuguucgugcagguag rno-miR-96 693 uuuggcacuagcacauuuuugcurno-miR-98 694 ugagguaguaaguuguauuguu rno-miR-99a 695aacccguagauccgaucuugug rno-miR-99b 696 cacccguagaaccgaccuugcgrno-miR-100 697 aacccguagauccgaacuugug rno-miR-101 698uacaguacugugauaacugaag rno-miR-103 699 agcagcauuguacagggcuaugarno-miR-106b 700 uaaagugcugacagugcagau rno-miR-107 701agcagcauuguacagggcuauca rno-miR-122a 702 uggagugugacaaugguguuugurno-miR-124a 703 uuaaggcacgcggugaaugcca rno-miR-125a 704ucccugagacccuuuaaccugug rno-miR-125b 705 ucccugagacccuaacuugugarno-miR-126* 706 cauuauuacuuuugguacgcg rno-miR-126 707ucguaccgugaguaauaaugc rno-miR-127 708 ucggauccgucugagcuuggcurno-miR-128a 709 ucacagugaaccggucucuuuu rno-miR-128b 710ucacagugaaccggucucuuuc rno-miR-130a 711 cagugcaauguuaaaagggcrno-miR-130b 712 cagugcaaugaugaaagggcau rno-miR-132 713uaacagucuacagccauggucg rno-miR-133a 714 uugguccccuucaaccagcugurno-miR-134 715 ugugacugguugaccagaggg rno-miR-135a 716uauggcuuuuuauuccuauguga rno-miR-136 717 acuccauuuguuuugaugauggarno-miR-137 718 uauugcuuaagaauacgcguag rno-miR-138 719 agcugguguugugaaucrno-miR-139 720 ucuacagugcacgugucu rno-miR-141 721 aacacugucugguaaagauggrno-miR-142-5p 722 cauaaaguagaaagcacuac rno-miR-142-3p 723uguaguguuuccuacuuuaugga rno-miR-143 724 ugagaugaagcacuguagcucarno-miR-144 725 uacaguauagaugauguacuag rno-miR-145 726guccaguuuucccaggaaucccuu rno-miR-146 727 ugagaacugaauuccauggguurno-miR-150 728 ucucccaacccuuguaccagug rno-miR-152 729ucagugcaugacagaacuugg rno-miR-153 730 uugcauagucacaaaaguga rno-miR-154731 uagguuauccguguugccuucg rno-miR-181c 732 aacauucaaccugucggugagurno-miR-181a 733 aacauucaacgcugucggugagu rno-miR-181b 734aacauucauugcugucgguggguu rno-miR-183 735 uauggcacugguagaauucacugrno-miR-184 736 uggacggagaacugauaagggu rno-miR-185 737uggagagaaaggcaguuc rno-miR-186 738 caaagaauucuccuuuugggcuu rno-miR-187739 ucgugucuuguguugcagccg rno-miR-190 740 ugauauguuugauauauuaggurno-miR-191 741 caacggaaucccaaaagcagcu rno-miR-192 742cugaccuaugaauugacagcc rno-miR-193 743 aacuggccuacaaagucccag rno-miR-194744 uguaacagcaacuccaugugga rno-miR-195 745 uagcagcacagaaauauuggcrno-miR-196 746 uagguaguuucauguuguugg rno-miR-199a 747cccaguguucagacuaccuguuc rno-miR-200c 748 aauacugccggguaaugauggarno-miR-200a 749 uaacacugucugguaacgaugu rno-miR-200b 750cucuaauacugccugguaaugaug rno-miR-203 751 gugaaauguuuaggaccacuagrno-miR-204 752 uucccuuugucauccuaugccu rno-miR-205 753uccuucauuccaccggagucug rno-miR-206 754 uggaauguaaggaaguguguggrno-miR-208 755 auaagacgagcaaaaagcuugu rno-miR-210 756cugugcgugugacagcggcug rno-miR-211 757 uucccuuugucauccuuugccu rno-miR-212758 uaacagucuccagucacggcc rno-miR-213 759 accaucgaccguugauuguaccrno-miR-214 760 acagcaggcacagacaggcag rno-miR-216 761uaaucucagcuggcaacugug rno-miR-217 762 uacugcaucaggaacugacuggaurno-miR-218 763 uugugcuugaucuaaccaugu rno-miR-219 764ugauuguccaaacgcaauucu rno-miR-221 765 agcuacauugucugcuggguuucrno-miR-222 766 agcuacaucuggcuacugggucuc rno-miR-223 767ugucaguuugucaaauacccc rno-miR-290 768 cucaaacuaugggggcacuuuuurno-miR-291-5p 769 caucaaaguggaggcccucucu rno-miR-291-3p 770aaagugcuuccacuuugugugcc rno-miR-292-5p 771 acucaaacugggggcucuuuugrno-miR-292-3p 772 aagugccgccagguuuugagugu rno-miR-296 773agggcccccccucaauccugu rno-miR-297 774 auguaugugugcauguaugcaugrno-miR-298 775 ggcagaggagggcuguucuucc rno-miR-299 776ugguuuaccgucccacauacau rno-miR-300 777 uaugcaagggcaagcucucuucrno-miR-320 778 aaaagcuggguugagagggcgaa rno-miR-321 779uaagccagggauuguggguuc

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications may be made withoutdeparting from the teachings herein. The foregoing examples are providedto better illustrate the disclosed teachings and are not intended tolimit the scope of the teachings herein.

We claim:
 1. A composition comprising: (a) a nucleic acid sample; (b) areverse transcriptase; (c) an unlabeled linker probe comprising a3′target-specific portion, a stem, and a loop; (d) at least one primer;and (e) a detector probe, wherein at least a portion of said detectorprobe corresponds with said stem of said linker probe, wherein saiddetector probe is a different molecule from said linker probe andwherein said detector probe comprises a detectable label.
 2. Thecomposition of claim 1, wherein said nucleic acid sample is an RNAsample.
 3. The composition of claim 1, further comprising at least onedNTP.
 4. The composition of claim 3, wherein said dNTP is a dUTP.
 5. Thecomposition of claim 1, wherein said linker probe is designed tohybridize to a target polynucleotide of interest and can extend to forma reaction product that includes the stem.
 6. The composition of claim5, wherein the target polynucleotide of interest is a miRNA.
 7. Thecomposition of claim 1, wherein said linker probe further comprises anidentifying portion.
 8. The composition of claim 1, further comprising aDNA polymerase.
 9. The composition of claim 6, wherein the compositioncomprises a forward primer specific for a miRNA and a universal reverseprimer.
 10. The composition of claim 9, wherein the universal reverseprimer comprises a nucleotide of the loop of the linker probe.
 11. Thecomposition of claim 10, wherein the nucleic acid sample is a celllysate.
 12. The composition of claim 6, wherein the 3′ target-specificportion is designed to hybridize to the 3′ end region of the miRNA. 13.The composition of claim 1, wherein the loop comprises 14-18nucleotides.
 14. The composition of claim 1, wherein the 3′ targetspecific portion comprises 5-8 nucleotides.
 15. The composition of claim1, wherein the detector probe comprises a universal base, a peptidenucleic acid (PNA), and/or a locked nucleic acid (LNA).
 16. Thecomposition of claim 1, wherein the detector probe comprises VIC or FAM.17. The composition of claim 1, wherein the detector probe is a5′-nuclease cleavable probe.
 18. The composition of claim 1, wherein thetarget polynucleotide of interest is 18-25 nucleotides in length.